Benzothiadiazepine compounds and their use as bile acid modulators

ABSTRACT

The invention relates to 1,2,5-benzothiadiazepine derivatives of formula (I). These compounds are bile acid modulators having apical sodium-dependent bile acid transporter (ASBT) and/or liver bile acid transport (LBAT) inhibitory activity. The invention also relates to pharmaceutical compositions comprising these compounds and to the use of these compounds in the treatment of cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases and liver diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Indian Application No. 201911049980,filed Dec. 4, 2019, the disclosure of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The invention relates to 1,2,5-benzothiadiazepine derivatives of formula(I). These compounds are bile acid modulators having apicalsodium-dependent bile acid transporter (ASBT) and/or liver bile acidtransport (LBAT) inhibitory activity. The invention also relates topharmaceutical compositions comprising these compounds and to the use ofthese compounds in the treatment of cardiovascular diseases, fatty acidmetabolism and glucose utilization disorders, gastrointestinal diseasesand liver diseases.

BACKGROUND

Bile acids are physiological detergents that play an important role inthe intestinal absorption and transport of lipids, nutrients andvitamins. They are also signaling molecules that activate nuclearreceptors and cell signaling pathways that regulate lipid, glucose andenergy metabolism. Bile acids are steroid acids that are synthesizedfrom cholesterol in the liver and stored in the gallbladder as mixedmicelles. During digestion, the duodenum triggers the release ofhormones that cause the gallbladder to contract, thereby releasing bileacids in the small intestine where they enable absorption of fat-solublevitamins and cholesterol. When they reach the ileum, bile acids arereabsorbed from the intestine and secreted into portal blood to returnto the liver via the portal venous circulation. Over 90% of the bileacids are thus recycled and returned to the liver. These bile acids arethen transported across the sinusoidal membrane of hepatocytes andre-secreted across the canalicular membrane into bile. In this firstpass, 75-90% of bile acids are taken up by hepatocytes, completing oneround of enterohepatic circulation. The fraction of bile acids thatescapes being cleared in the liver enters the systemic circulation wherethe free bile acids are filtered by the renal glomerulus, efficientlyreclaimed in the proximal tubules and exported back into the systemiccirculation. Interestingly, most of the bile acids secreted across thecanalicular membrane into bile are derived from the recirculating poolwith less than 10% coming from new de novo hepatic synthesis. The smallfraction of bile acids that is not reabsorbed in the ileum reaches thecolon. Within the intestinal lumen, the primary bile acids aretransformed into secondary bile acids under the action of intestinalbacteria, mainly by single or dual dehydroxylation reactions of thesteroid nucleus. The bile acids that escape intestinal absorption arethereafter excreted into the faeces.

Overall, the efficient transport system helps maintain a constant bileacid pool, ensuring sufficiently high levels of conjugated bile acids inthe intestine to promote lipid absorption as well as reduce the smallintestinal bacterial load. The system also minimizes fecal and urinarybile acid loss and protects the intestinal and hepatobiliarycompartments by eliminating potentially cytotoxic detergents (asreviewed by Kosters and Karpen (Xenobiotica 2008, vol. 38, p.1043-1071); by Chiang (J. Lipid Res. 2009, vol. 50, p. 1955-1966); andby Dawson (Handb. Exp. Pharmacol. 2011, vol. 201, p. 169-203)).

The regulation of the bile acid pool size has been found to play a keyrole in cholesterol homeostasis by hepatic conversion of cholesterol tobile acid, which represents a major route for elimination of cholesterolfrom the body. The liver plays an essential role in removing endogenousand xenobiotic compounds from the body. The normal hepatobiliarysecretion and enterohepatic circulation are required for the eliminationof endogenous compounds such as cholesterol and bilirubin and theirmetabolites from the body, thereby maintaining lipid and bile acidhomeostasis. (Kosters and Karpen, Xenobiotica 2008, vol. 38, p.1043-1071).

The reabsorption of bile acids in the ileum may be inhibited by apicalsodium-dependent bile acid transporter (ASBT) inhibitor compounds.Inhibition of bile acid reabsorption has been reported useful in thetreatment of several diseases, including dyslipidemia, diabetes,obesity, constipation, cholestatic liver diseases, non-alcoholicsteatohepatitis and other hepatic diseases. A number of ASBT inhibitorcompounds has been disclosed over the past decades, see e.g. WO93/16055, WO 94/18183, WO 94/18184, WO 96/05188, WO 96/08484, WO96/16051, WO 97/33882, WO 98/03818, WO 98/07449, WO 98/40375, WO99/35135, WO 99/64409, WO 99/64410, WO 00/47568, WO 00/61568, WO00/38725, WO 00/38726, WO 00/38727, WO 00/38728, WO 00/38729, WO01/66533, WO 01/68096, WO 02/32428, WO 02/50051, WO 03/020710, WO03/022286, WO 03/022825, WO 03/022830, WO 03/061663, WO 03/091232, WO03/106482, WO 2004/006899, WO 2004/076430, WO 2007/009655, WO2007/009656, WO 2011/137135, WO 2019/234077, WO 2020/161216, WO2020/161217, DE 19825804, EP 864582, EP 489423, EP549967, EP573848,EP624593, EP624594, EP624595, EP624596, EP0864582, EP1173205, EP1535913and EP3210977.

Despite the number of ASBT inhibitor compounds that have been previouslyreported, there is a need for additional bile acid modulating compoundsthat have an optimized profile with respect to potency, selectivity andbioavailability.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that certain 1,2,5-benzothiadiazepine derivatesare potent inhibitors of apical sodium-dependent bile acid transporter(ASBT) and/or liver bile acid transporter (LBAT), and may be useful fortreating diseases wherein inhibition of bile acid circulation isdesirable.

In a first aspect, the invention relates to a compound of formula (I)

wherein

-   -   R¹ and R² are each independently C₁₋₄ alkyl;    -   R³ is independently selected from the group consisting of        hydrogen, halogen, hydroxy, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄        alkoxy, C₁₋₄ haloalkoxy, cyano, nitro, amino, N—(C₁₋₄        alkyl)amino, N,N-di(C₁₋₄ alkyl)amino, and N-(aryl-C₁₋₄        alkyl)amino;    -   n is an integer 1, 2 or 3; and    -   R⁴ is selected from the group consisting of hydrogen, halogen,        hydroxy, cyano, C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₁₋₄ alkoxy, C₃₋₆        cycloalkyloxy, C₁₋₄ alkylthio, C₃₋₆ cycloalkylthio, amino,        N—(C₁₋₄ alkyl)-amino and N,N-di(C₁₋₄ alkyl)amino;    -   or a pharmaceutically acceptable salt thereof,

with the proviso that the compound is not a compound from the groupconsisting of:

-   2-((3,3-dibutyl-7-methyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid;-   2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid; and-   2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid.

In some embodiments, R¹ is n-butyl.

In some embodiments, R² is C₂₋₄ alkyl. In a preferred embodiment, R² isethyl. In another preferred embodiment, R² is n-propyl. In yet anotherpreferred embodiment, R² is n-butyl.

In some embodiments, R³ is independently selected from the groupconsisting of hydrogen, halogen, hydroxy, cyano, C₁₋₄ haloalkyl, C₁₋₄alkoxy and C₁₋₄ haloalkoxy. In a preferred embodiment, R³ isindependently selected from the group consisting of hydrogen, fluoro,chloro, bromo, hydroxy, cyano, trifluoromethyl, methoxy andtrifluoromethoxy.

In a preferred embodiment, n is 1, i.e. the phenyl-ring is substitutedwith only one substituent R³. In another preferred embodiment, R³ is inthe para-position.

In some embodiments, R⁴ is selected from the group consisting ofhalogen, hydroxy, cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylthio, amino,N—(C₁₋₄ alkyl)amino and N,N-di(C₁₋₄ alkyl)amino. In a preferredembodiment, R⁴ is selected from the group consisting of fluoro, chloro,bromo, hydroxy, cyano, methyl, methoxy, ethoxy, methylthio, ethylthio,amino, methylamino and dimethylamino. In another preferred embodiment,R⁴ is selected from the group consisting of fluoro, chloro, bromo,hydroxy, cyano, methoxy, ethoxy, methylthio, ethylthio anddimethylamino.

In a preferred embodiment, the compound of formula (I) is a compound offormula (I-a)

wherein

-   -   R² is C₂₋₄ alkyl;    -   R³ is independently selected from the group consisting of        hydrogen, halogen, hydroxy, cyano, C₁₋₄ haloalkyl, C₁₋₄ alkoxy        and C₁₋₄ haloalkoxy;    -   n is an integer 1 or 2;    -   R⁴ is selected from the group consisting of halogen, hydroxy,        cyano, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylthio, amino, N—(C₁₋₄        alkyl)amino and N,N-di(C₁₋₄ alkyl)amino; or a pharmaceutically        acceptable salt thereof,

with the proviso that the compound is not a compound from the groupconsisting of:

-   2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid; and-   2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid.

In another preferred embodiment, the compound of formula (I) is acompound of formula (I-b)

wherein

-   -   R² is ethyl, n-propyl or n-butyl;    -   R³ is selected from the group consisting of hydrogen, fluoro,        chloro, bromo, hydroxy, cyano, trifluoromethyl, methoxy, and        trifluoromethoxy;    -   R⁴ is selected from the group consisting of fluoro, chloro,        bromo, hydroxy, cyano, methoxy, ethoxy, methylthio, ethylthio        and dimethylamino;

or a pharmaceutically acceptable salt thereof,

with the proviso that the compound is not a compound from the groupconsisting of:

-   2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid; and-   2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid.

Preferred compounds of the invention are compounds of formula (I-b), asdefined above, wherein M and R¹ to R⁴ are as indicated in Table 1 below,or a pharmaceutically acceptable salt thereof:

TABLE 1 R¹ R² R³ CH₂CH₃ H SCH₃ CH₂CH₃ F SCH₃ CH₂CH₃ OCH₃ SCH₃ CH₂CH₃ OHSCH₃ CH₂CH₃ Cl SCH₃ CH₂CH₃ CN SCH₃ CH₂CH₃ CF₃ SCH₃ CH₂CH₃ H N(CH₃)₂CH₂CH₃ F N(CH₃)₂ CH₂CH₃ OCH₃ N(CH₃)₂ CH₂CH₃ OH N(CH₃)₂ CH₂CH₃ Cl N(CH₃)₂CH₂CH₃ CN N(CH₃)₂ CH₂CH₃ CF₃ N(CH₃)₂ CH₂CH₃ H SCH₂CH₃ CH₂CH₃ F SCH₂CH₃CH₂CH₃ OCH₃ SCH₂CH₃ CH₂CH₃ OH SCH₂CH₃ CH₂CH₃ Cl SCH₂CH₃ CH₂CH₃ CNSCH₂CH₃ CH₂CH₃ CF₃ SCH₂CH₃ CH₂CH₂CH₃ H SCH₃ CH₂CH₂CH₃ F SCH₃ CH₂CH₂CH₃OCH₃ SCH₃ CH₂CH₂CH₃ OH SCH₃ CH₂CH₂CH₃ Cl SCH₃ CH₂CH₂CH₃ CN SCH₃CH₂CH₂CH₃ CF₃ SCH₃ CH₂CH₂CH₃ H N(CH₃)₂ CH₂CH₂CH₃ F N(CH₃)₂ CH₂CH₂CH₃OCH₃ N(CH₃)₂ CH₂CH₂CH₃ OH N(CH₃)₂ CH₂CH₂CH₃ Cl N(CH₃)₂ CH₂CH₂CH₃ CNN(CH₃)₂ CH₂CH₂CH₃ CF₃ N(CH₃)₂ CH₂CH₂CH₃ H SCH₂CH₃ CH₂CH₂CH₃ F SCH₂CH₃CH₂CH₂CH₃ OCH₃ SCH₂CH₃ CH₂CH₂CH₃ OH SCH₂CH₃ CH₂CH₂CH₃ Cl SCH₂CH₃CH₂CH₂CH₃ CN SCH₂CH₃ CH₂CH₂CH₃ CF₃ SCH₂CH₃ CH₂CH₂CH₂CH₃ F SCH₃CH₂CH₂CH₂CH₃ OCH₃ SCH₃ CH₂CH₂CH₂CH₃ OH SCH₃ CH₂CH₂CH₂CH₃ Cl SCH₃CH₂CH₂CH₂CH₃ CN SCH₃ CH₂CH₂CH₂CH₃ CF₃ SCH₃ CH₂CH₂CH₂CH₃ H N(CH₃)₂CH₂CH₂CH₂CH₃ F N(CH₃)₂ CH₂CH₂CH₂CH₃ OCH₃ N(CH₃)₂ CH₂CH₂CH₂CH₃ OH N(CH₃)₂CH₂CH₂CH₂CH₃ Cl N(CH₃)₂ CH₂CH₂CH₂CH₃ CN N(CH₃)₂ CH₂CH₂CH₂CH₃ CF₃ N(CH₃)₂CH₂CH₂CH₂CH₃ H SCH₂CH₃ CH₂CH₂CH₂CH₃ F SCH₂CH₃ CH₂CH₂CH₂CH₃ OCH₃ SCH₂CH₃CH₂CH₂CH₂CH₃ OH SCH₂CH₃ CH₂CH₂CH₂CH₃ Cl SCH₂CH₃ CH₂CH₂CH₂CH₃ CN SCH₂CH₃CH₂CH₂CH₂CH₃ CF₃ SCH₂CH₃

In a particular embodiment, the compound of formula (I) is selected fromthe group consisting of:

-   2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiadiazepin-8-yl)oxy)acetic    acid;-   (S)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid; and-   (R)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetic    acid;

or a pharmaceutically acceptable salt thereof.

As used herein, the term “halo” refers to fluoro, chloro, bromo andiodo.

As used herein, the term “C₁₋₆ alkyl” refers to a straight or branchedalkyl group having from 1 to 6 carbon atoms, and the term “C₁₋₄ alkyl”refers to a straight or branched alkyl group having from 1 to 4 carbonatoms. Examples of C₁₋₄ alkyl include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

As used herein, the term “C₁₋₄ haloalkyl” refers to a straight orbranched C₁₋₄ alkyl group, as defined herein, wherein one or morehydrogen atoms have been replaced with halogen. Examples of C₁₋₄haloalkyl include chloromethyl, fluoroethyl and trifluoromethyl.

As used herein, the terms “C₁₋₄ alkoxy” and “C₁₋₄ alkylthio” refer to astraight or branched C₁₋₄ alkyl group attached to the remainder of themolecule through an oxygen or sulphur atom, respectively.

As used herein, the term “C₃₋₆ cycloalkyl” refers to a monocyclicsaturated hydrocarbon ring having from 3 to 6 carbon atoms. Examples ofC₃₋₆ cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

The term “aryl” denotes an aromatic monocyclic ring composed of 6 carbonatoms or an aromatic bicyclic ring system composed of 10 carbon atoms.Examples of aryl include phenyl, naphthyl and azulenyl.

The term “amino” refers to an —NH₂ group. As used herein, the terms“N—(C₁₋₄ alkyl)amino” and “N,N-di(C₁₋₄ alkyl)amino” refer to an aminogroup wherein one or both hydrogen atom(s), respectively, are replacedwith a straight or branched C₁₋₄ alkyl group. Examples of N—(C₁₋₄alkyl)amino include methylamino, ethylamino and tert-butylamino, andexamples of N,N-di-(C₁₋₄ alkyl)amino include dimethylamino anddiethylamino.

As used herein, the term “N-(aryl-C₁₋₄ alkyl)amino” refers to an aminogroup wherein a hydrogen atom is replaced with an aryl-C₁₋₄ alkyl group.Examples of N-(aryl-C₁₋₄ alkyl)amino include benzylamino andphenylethylamino.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions and/or dosage forms that are suitablefor human pharmaceutical use and that are generally safe, non-toxic andneither biologically nor otherwise undesirable.

As used herein, the term “about” refers to a value or parameter hereinthat includes (and describes) embodiments that are directed to thatvalue or parameter per se. For example, description referring to “about20” includes description of “20.” Numeric ranges are inclusive of thenumbers defining the range. Generally speaking, the term “about” refersto the indicated value of the variable and to all values of the variablethat are within the experimental error of the indicated value (e.g.,within the 95% confidence interval for the mean) or within 10 percent ofthe indicated value, whichever is greater.

The 1,2,5-benzothiadiazepine compounds of formula (I), orpharmaceutically acceptable salts thereof, are inhibitors of the apicalsodium-dependent bile acid transporter (ASBT inhibitors), of the liverbile acid transporter (LBAT inhibitors), or of both the apicalsodium-dependent bile acid and liver bile acid transporters (dualASBT/LBAT inhibitors). They are therefore useful in the treatment orprevention of conditions, disorders and diseases wherein inhibition ofbile acid circulation is desirable, such as cardiovascular diseases,fatty acid metabolism and glucose utilization disorders,gastrointestinal diseases and liver diseases.

Cardiovascular diseases and disorders of fatty acid metabolism andglucose utilization include, but are not limited to,hypercholesterolemia; disorders of fatty acid metabolism; type 1 andtype 2 diabetes mellitus; complications of diabetes, includingcataracts, micro- and macrovascular diseases, retinopathy, neuropathy,nephropathy and delayed wound healing, tissue ischaemia, diabetic foot,arteriosclerosis, myocardial infarction, acute coronary syndrome,unstable angina pectoris, stable angina pectoris, stroke, peripheralarterial occlusive disease, cardiomyopathy, heart failure, heart rhythmdisorders and vascular restenosis; diabetes-related diseases such asinsulin resistance (impaired glucose homeostasis), hyperglycemia,hyperinsulinemia, elevated blood levels of fatty acids or glycerol,obesity, dyslipidemia, hyperlipidemia including hypertriglyceridemia,metabolic syndrome (syndrome X), atherosclerosis and hypertension; andfor increasing high density lipoprotein levels.

Gastrointestinal diseases and disorders include constipation (includingchronic constipation, functional constipation, chronic idiopathicconstipation (CIC), intermittent/sporadic constipation, constipationsecondary to diabetes mellitus, constipation secondary to stroke,constipation secondary to chronic kidney disease, constipation secondaryto multiple sclerosis, constipation secondary to Parkinson's disease,constipation secondary to systemic sclerosis, drug induced constipation,irritable bowel syndrome with constipation (IBS-C), irritable bowelsyndrome mixed (IBS-M), pediatric functional constipation and opioidinduced constipation); Crohn's disease; primary bile acid malabsorption;irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ilealinflammation; and reflux disease and complications thereof, such asBarrett's esophagus, bile reflux esophagitis and bile reflux gastritis.

A liver disease as defined herein is any disease in the liver and inorgans connected therewith, such as the pancreas, portal vein, the liverparenchyma, the intrahepatic biliary tree, the extrahepatic biliarytree, and the gall bladder. In some cases, a liver disease a bileacid-dependent liver disease. Liver diseases and disorders include, butare not limited to, an inherited metabolic disorder of the liver; inbornerrors of bile acid synthesis; congenital bile duct anomalies; biliaryatresia; post-Kasai biliary atresia; post-liver transplantation biliaryatresia; neonatal hepatitis; neonatal cholestasis; hereditary forms ofcholestasis; cerebrotendinous xanthomatosis; a secondary defect of BAsynthesis; Zellweger's syndrome; cystic fibrosis-associated liverdisease; alpha1-antitrypsin deficiency; Alagilles syndrome (ALGS); Bylersyndrome; a primary defect of bile acid (BA) synthesis; progressivefamilial intrahepatic cholestasis (PFIC) including PFIC-1, PFIC-2,PFIC-3 and non-specified PFIC, post-biliary diversion PFIC andpost-liver transplant PFIC; benign recurrent intrahepatic cholestasis(BRIC) including BRIC1, BRIC2 and non-specified BRIC, post-biliarydiversion BRIC and post-liver transplant BRIC; autoimmune hepatitis;primary biliary cirrhosis (PBC); liver fibrosis; non-alcoholic fattyliver disease (NAFLD); non-alcoholic steatohepatitis (NASH); portalhypertension; cholestasis; Down syndrome cholestasis; drug-inducedcholestasis; intrahepatic cholestasis of pregnancy (jaundice duringpregnancy); intrahepatic cholestasis; extrahepatic cholestasis;parenteral nutrition associated cholestasis (PNAC); lowphospholipid-associated cholestasis; lymphedema cholestasis syndrome 1(LSC1); primary sclerosing cholangitis (PSC); immunoglobulin G4associated cholangitis; primary biliary cholangitis; cholelithiasis(gall stones); biliary lithiasis; choledocholithiasis; gallstonepancreatitis; Caroli disease; malignancy of bile ducts; malignancycausing obstruction of the biliary tree; biliary strictures; AIDScholangiopathy; ischemic cholangiopathy; pruritus due to cholestasis orjaundice; pancreatitis; chronic autoimmune liver disease leading toprogressive cholestasis; hepatic steatosis; alcoholic hepatitis; acutefatty liver; fatty liver of pregnancy; drug-induced hepatitis; ironoverload disorders; congenital bile acid synthesis defect type 1 (BAStype 1); drug-induced liver injury (DILI); hepatic fibrosis; congenitalhepatic fibrosis; hepatic cirrhosis; Langerhans cell histiocytosis(LCH); neonatal ichthyosis sclerosing cholangitis (NISCH);erythropoietic protoporphyria (EPP); idiopathic adulthood ductopenia(IAD); idiopathic neonatal hepatitis (INH); non syndromic paucity ofinterlobular bile ducts (NS PILBD); North American Indian childhoodcirrhosis (NAIC); hepatic sarcoidosis; amyloidosis; necrotizingenterocolitis; serum bile acid-caused toxicities, including cardiacrhythm disturbances (e.g., atrial fibrillation) in setting of abnormalserum bile acid profile, cardiomyopathy associated with liver cirrhosis(“cholecardia”), and skeletal muscle wasting associated with cholestaticliver disease; polycystic liver disease; viral hepatitis (includinghepatitis A, hepatitis B, hepatitis C, hepatitis D and hepatitis E);hepatocellular carcinoma (hepatoma); cholangiocarcinoma; bileacid-related gastrointestinal cancers; and cholestasis caused by tumoursand neoplasms of the liver, of the biliary tract and of the pancreas.Compounds of formula (I), or pharmaceutically acceptable salts thereof,are also useful in the enhancement of corticosteroid therapy in liverdisease.

Other diseases that may be treated or prevented by the compounds offormula (I), or pharmaceutically acceptable salts thereof, includehyperabsorption syndromes (including abetalipoproteinemia, familialhypobetalipoproteinemia (FHBL), chylomicron retention disease (CRD) andsitosterolemia); hypervitaminosis and osteopetrosis; hypertension;glomerular hyperfiltration; polycystic kidney disease (PKD), includingautosomal dominant polycystic kidney disease (ADPKD) and autosomalrecessive polycystic kidney disease (ARPKD); and pruritus of renalfailure. The compounds are also useful in the protection against liver-or metabolic disease-associated kidney injury.

The transport of bile acids in the human body is controlled by theaction of the members of the SLC10 family of solute carrier proteins, inparticular by the Na⁺-taurocholate cotransporting polypeptide (NTCP,also called liver bile acid transporter (LBAT); gene symbol SLC10A1),which is expressed in the sinusoidal membrane of hepatocytes, and by theapical sodium dependent bile acid transporter (ASBT, also called ilealbile acid transporter (IBAT), ISBT, ABAT or NTCP2; gene symbol SLC10A2),which is expressed in the apical membrane of ileal enterocytes, proximalrenal tubule cells, biliary epithelium, large cholangiocytes andgallbladder epithelial cells. In the liver, bile acids are efficientlyextracted from portal blood by the liver bile acid transporter (LBAT)and re-secreted across the canalicular membrane by the bile salt exportpump (BSEP; gene symbol ABCB11). The reabsorption of bile acids in theileum is handled by the apical sodium-dependent bile acid transporter(ASBT), where it is commonly referred to as ileal bile acid transporter(IBAT). Both LBAT and ASBT function as electrogenic sodium-solutecotransporters that move two or more Na⁺ ions per molecule of solute.

Xenobiotics and endobiotics, including bile acids, are taken up by theliver from portal blood and secreted into bile by distinct transportproteins with individualized substrate specificities. Glycine- andtaurine-conjugated bile acids exist in anionic form and are unable tocross membranes by diffusion, and thus, are completely dependent onmembrane transport proteins to enter or exit the hepatocyte (Kosters andKarpen, Xenobiotica 2008, vol. 38, p. 1043-1071). ASBT and LBAT preferglycine- and taurine-conjugated bile salts over their unconjugatedcounterparts and demonstrate a higher affinity for dihydroxy bile saltsthan for trihydroxy bile salts. No non-bile acid substrates have beenidentified for ASBT yet, however, LBAT was also found to transport avariety of steroid sulfates, hormones and xenobiotics.

LBAT is not as thoroughly characterized as ASBT in terms of druginhibition requirements. Dong et al. have identified FDA approved drugsthat inhibit human LBAT and compared LBAT and ASBT inhibitionrequirements. A series of LBAT inhibition studies were performed usingFDA approved drugs, in concert with iterative computational modeldevelopment. Screening studies identified 27 drugs as novel LBATinhibitors, including irbesartan (Ki=11.9 μM) and ezetimibe (Ki=25.0μM). The common feature pharmacophore indicated that two hydrophobes andone hydrogen bond acceptor were important for inhibition of LBAT. From72 drugs screened in vitro, a total of 31 drugs inhibited LBAT, while 51drugs (i.e. more than half) inhibited ASBT. Hence, while there wasinhibitor overlap, ASBT unexpectedly was more permissive to druginhibition than was LBAT, and this may be related to LBAT's possessingfewer pharmacophore features (Dong et al., Mol. Pharm. 2013, vol. 10, p.1008-1019).

Vaz et al. describe the identification of LBAT deficiency as a newinborn error of metabolism with a relatively mild clinical phenotype.The identification of LBAT deficiency confirms that this transporter isthe main import system for conjugated bile salts into the liver, butalso indicates that auxiliary transporters are able to sustain theenterohepatic cycle in its absence (Vaz et al., Hepatology 2015, vol.61, p. 260-267). These findings support the hypothesis that LBATinhibition is a safe mechanism of action, as the hepatocytes still havethe possibility to take up the necessary amount of bile acids.

Liu et al. describe the identification of a new type of hypercholanemiathat is associated with homozygosity for the p.Ser267Phe mutation inSLC10A1 (LBAT). The allele frequency of this mutation in gene SLC10A1varies in different populations, with the highest incidence occurring inSouthern China (8% and 12% in Chinese Han and Dai respectively) and inVietnam (11%). This “hidden” hypercholanemia was believed to affect0.64% of the Southern Han, 1.44% of the Dai Chinese population, and1.21% of the Vietnamese population. An increase in conjugated andunconjugated serum BA levels in the homozygous individuals was alsoobserved. Liu et al. suggest that this finding is most likely due toreduced BA transport from the portal circulation into hepatocytes. Thissupports the hypothesis that the physiological function of theenterohepatic circulation is not only to recycle bile acids but also toclear bile acids from the circulation to achieve homeostasis (Karpen andDawson, Hepatology 2015, vol. 61, p. 24-27). Alternatively, the livermay synthesize increased levels of bile acids to compensate for thereduced enterohepatic recirculation in the homozygous carriers. As LBATalso transports unconjugated bile acids, the increase of theunconjugated bile acids in this study was not surprising (Liu et al.,Scientific Reports 2017, 7: 9214, p. 1-7).

LBAT has been found to be downregulated in several forms of cholestaticliver injury and cholestasis, whereas ASBT has been found to bedownregulated in a variety of gastrointestinal disorders such as Crohn'sdisease, primary bile acid malabsorption, inflammatory bowel disease,and ileal inflammation but upregulated in cholestasis. LBAT alsofunctions as a cellular receptor for viral entry of the hepatitis Bvirus (HBV) and hepatitis D virus (HDV), which in turn is the majorcause of liver disease and hepatocellular carcinoma.

ASBT inhibition has been investigated for decreasing plasma cholesterollevels and improving insulin resistance, as well as to relieving thehepatic bile acid burden in cholestatic liver disease. In addition, ASBTinhibition has been found to restore insulin levels and normoglycemia,thus establishing ASBT inhibition as a promising treatment for type 2diabetes mellitus. ASBT inhibitors are also used for treatment offunctional constipation.

As ASBT is predominantly expressed in the ileum (where it is oftenreferred to as IBAT), ASBT inhibitors need not be systemicallyavailable. On the other hand, ASBT is also expressed in the proximaltubule cells of the kidneys. ASBT inhibitors that are systemicallyavailable may therefore also inhibit the reuptake of bile acids in thekidneys. It is believed that this would lead to increased levels of bileacids in urine, and to an increased removal of bile acids from the bodyvia the urine. Systemically available ASBT inhibitors that exert theireffect not only in the ileum but also in the kidneys are thereforeexpected to lead to a greater reduction of bile acid levels thannon-systemically available ASBT inhibitors that only exert their effectin the ileum.

Compounds having a high ASBT inhibiting potency are particularlysuitable for the treatment of liver diseases that cause cholestasis,such as progressive familial intrahepatic cholestasis (PFIC), Alagillessyndrome, biliary atresia and non-alcoholic steatohepatitis (NASH).

Biliary atresia is a rare pediatric liver disease that involves apartial or total blockage (or even absence) of large bile ducts. Thisblockage or absence causes cholestasis that leads to the accumulation ofbile acids that damages the liver. In some embodiments, the accumulationof bile acids occurs in the extrahepatic biliary tree. In someembodiments, the accumulation of bile acids occurs in the intrahepaticbiliary tree. The current standard of care is the Kasai procedure, whichis a surgery that removes the blocked bile ducts and directly connects aportion of the small intestine to the liver. There are currently noapproved drug therapies for this disorder.

Provided herein are methods for treating biliary atresia in a subject inneed thereof, the methods comprising administration of a therapeuticallyeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the subject has undergonethe Kasai procedure prior to administration of a compound of formula(I), or a pharmaceutically acceptable salt thereof. In some embodiments,the subject is administered a compound of formula (I), or apharmaceutically acceptable salt thereof, prior to undergoing the Kasaiprocedure. In some embodiments, the treatment of biliary atresiadecreases the level of serum bile acids in the subject. In someembodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of a compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, thetreatment of bilary atresia includes treatment of pruritus.

PFIC is a rare genetic disorder that is estimated to affect between onein every 50,000 to 100,000 children born worldwide and causesprogressive, life-threatening liver disease.

One manifestation of PFIC is pruritus, which often results in a severelydiminished quality of life. In some cases, PFIC leads to cirrhosis andliver failure. Current therapies include Partial External BiliaryDiversion (PEBD) and liver transplantation, however, these options cancarry substantial risk of post-surgical complications, as well aspsychological and social issues.

Three alternative gene defects have been identified that correlate tothree separate PFIC subtypes known as types 1, 2 and 3:

-   -   PFIC, type 1, which is sometimes referred to as “Byler disease,”        is caused by impaired bile secretion due to mutations in the        ATP8B1 gene, which codes for a protein that helps to maintain an        appropriate balance of fats known as phospholipids in cell        membranes in the bile ducts. An imbalance in these phospholipids        is associated with cholestasis and elevated bile acids in the        liver. Subjects affected by PFIC, type 1 usually develop        cholestasis in the first months of life and, in the absence of        surgical treatment, progress to cirrhosis and end-stage liver        disease before the end of the first decade of life.    -   PFIC, type 2, which is sometimes referred to as “Byler        syndrome,” is caused by impaired bile salt secretion due to        mutations in the ABCB11 gene, which codes for a protein, known        as the bile salt export pump, that moves bile acids out of the        liver. Subjects with PFIC, type 2 often develop liver failure        within the first few years of life and are at increased risk of        developing a type of liver cancer known as hepatocellular        carcinoma.    -   PFIC, type 3, which typically presents in the first years of        childhood with progressive cholestasis, is caused by mutations        in the ABCB4 gene, which codes for a transporter that moves        phospholipids across cell membranes.

In addition, TJP2 gene, NR1H4 gene or Myo5b gene mutations have beenproposed to be causes of PFIC. In addition, some subjects with PFIC donot have a mutation in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 orMyo5b genes. In these cases, the cause of the condition is unknown.

Exemplary mutations of the ATP8B1 gene or the resulting protein arelisted in Tables 2 and 3, with numbering based on the human wild typeATP8B1 protein (e.g., SEQ ID NO: 1) or gene (e.g., SEQ ID NO: 2).Exemplary mutations of the ABCB11 gene or the resulting protein arelisted in Tables 4 and 5, with numbering based on the human wild typeABCB11 protein (e.g., SEQ ID NO: 3) or gene (e.g., SEQ ID NO: 4).

As can be appreciated by those skilled in the art, an amino acidposition in a reference protein sequence that corresponds to a specificamino acid position in SEQ ID NO: 1 or 3 can be determined by aligningthe reference protein sequence with SEQ ID NO: 1 or 3 (e.g., using asoftware program, such as ClustalW2). Changes to these residues(referred to herein as “mutations”) may include single or multiple aminoacid substitutions, insertions within or flanking the sequences, anddeletions within or flanking the sequences. As can be appreciated bythose skilled in the art, an nucleotide position in a reference genesequence that corresponds to a specific nucleotide position in SEQ IDNO: 2 or 4 can be determined by aligning the reference gene sequencewith SEQ ID NO: 2 or 4 (e.g., using a software program, such asClustalW2). Changes to these residues (referred to herein as“mutations”) may include single or multiple nucleotide substitutions,insertions within or flanking the sequences, and deletions within orflanking the sequences. See also Kooistra, et al., “KLIFS: A structuralkinase-ligand interaction database,” Nucleic Acids Res. 2016, vol. 44,no. D1, pp. D365-D371, which is incorporated by reference in itsentirety herein.

Canonical protein sequence of ATP8B1 - Uniprot ID O43520 (SEQ ID NO: 1)MSTERDSETT FDEDSQPNDE VVPYSDDETE DELDDQGSAV EPEQNRVNRE AEENREPFRKECTWQVKAND RKYHEQPHFM NTKFLCIKES KYANNAIKTY KYNAFTFIPM NLFEQFKRAANLYFLALLIL QAVPQISTLA WYTTLVPLLV VLGVTAIKDL VDDVARHKMD KEINNRTCEVIKDGRFKVAK WKEIQVGDVI RLKKNDFVPA DILLLSSSEP NSLCYVETAE LDGETNLKFKMSLEITDQYL QREDTLATFD GFIECEEPNN RLDKFTGTLF WRNTSFPLDA DKILLRGCVIRNTDFCHGLV IFAGADTKIM KNSGKTRFKR TKIDYLMNYM VYTIFVVLIL LSAGLAIGHAYWEAQVGNSS WYLYDGEDDT PSYRGFLIFW GYIIVLNTMV PISLYVSVEV IRLGQSHFINWDLQMYYAEK DTPAKARTTT LNEQLGQIHY IFSDKTGTLT QNIMTFKKCC INGQIYGDHRDASQHNHNKI EQVDFSWNTY ADGKLAFYDH YLIEQIQSGK EPEVRQFFFL LAVCHTVMVDRTDGQLNYQA ASPDEGALVN AARNFGFAFL ARTQNTITIS ELGTERTYNV LAILDFNSDRKRMSIIVRTP EGNIKLYCKG ADTVIYERLH RMNPTKQETQ DALDIFANET LRTLCLCYKEIEEKEFTEWN KKFMAASVAS TNRDEALDKV YEEIEKDLIL LGATAIEDKL QDGVPETISKLAKADIKIWV LTGDKKETAE NIGFACELLT EDTTICYGED INSLLHARME NQRNRGGVYAKFAPPVQESF FPPGGNRALI ITGSWLNEIL LEKKTKRNKI LKLKFPRTEE ERRMRTQSKRRLEAKKEQRQ KNFVDLACEC SAVICCRVTP KQKAMVVDLV KRYKKAITLA IGDGANDVNMIKTAHIGVGI SGQEGMQAVM SSDYSFAQFR YLQRLLLVHG RWSYIRMCKF LRYFFYKNFAFTLVHFWYSF FNGYSAQTAY EDWFITLYNV LYTSLPVLLM GLLDQDVSDK LSLRFPGLYIVGQRDLLFNY KRFFVSLLHG VLTSMILFFI PLGAYLQTVG QDGEAPSDYQ SFAVTIASALVITVNFQIGL DTSYWTFVNA FSIFGSIALY FGIMFDFHSA GIHVLFPSAF QFTGTASNALRQPYIWLTII LAVAVCLLPV VAIRFLSMTI WPSESDKIQK HRKRLKAEEQ WQRRQQVFRRGVSTRRSAYA FSHQRGYADL ISSGRSIRKK RSPLDAIVAD GTAEYRRTGD SCanonical DNA Sequence for ATP8B1 (SEQ ID NO: 2)ATG AGT ACA GAA AGA GAC TCA GAA ACG ACA TTT GAC GAG GAT TCT CAG CCTAAT GAC GAA GTG GTT CCC TAC AGT GAT GAT GAA ACA GAA GAT GAA CTT GATGAC CAG GGG TCT GCT GTT GAA CCA GAA CAA AAC CGA GTC AAC AGG GAA GCAGAG GAG AAC CGG GAG CCA TTC AGA AAA GAA TGT ACA TGG CAA GTC AAA GCAAAC GAT CGC AAG TAC CAC GAA CAA CCT CAC TTT ATG AAC ACA AAA TTC TTGTGT ATT AAG GAG AGT AAA TAT GCG AAT AAT GCA ATT AAA ACA TAC AAG TACAAC GCA TTT ACC TTT ATA CCA ATG AAT CTG TTT GAG CAG TTT AAG AGA GCAGCC AAT TTA TAT TTC CTG GCT CTT CTT ATC TTA CAG GCA GTT CCT CAA ATCTCT ACC CTG GCT TGG TAC ACC ACA CTA GTG CCC CTG CTT GTG GTG CTG GGCGTC ACT GCA ATC AAA GAC CTG GTG GAC GAT GTG GCT CGC CAT AAA ATG GATAAG GAA ATC AAC AAT AGG ACG TGT GAA GTC ATT AAG GAT GGC AGG TTC AAAGTT GCT AAG TGG AAA GAA ATT CAA GTT GGA GAC GTC ATT CGT CTG AAA AAAAAT GAT TTT GTT CCA GCT GAC ATT CTC CTG CTG TCT AGC TCT GAG CCT AACAGC CTC TGC TAT GTG GAA ACA GCA GAA CTG GAT GGA GAA ACC AAT TTA AAATTT AAG ATG TCA CTT GAA ATC ACA GAC CAG TAC CTC CAA AGA GAA GAT ACATTG GCT ACA TTT GAT GGT TTT ATT GAA TGT GAA GAA CCC AAT AAC AGA CTAGAT AAG TTT ACA GGA ACA CTA TTT TGG AGA AAC ACA AGT TTT CCT TTG GATGCT GAT AAA ATT TTG TTA CGT GGC TGT GTA ATT AGG AAC ACC GAT TTC TGCCAC GGC TTA GTC ATT TTT GCA GGT GCT GAC ACT AAA ATA ATG AAG AAT AGTGGG AAA ACC AGA TTT AAA AGA ACT AAA ATT GAT TAC TTG ATG AAC TAC ATGGTT TAC ACG ATC TTT GTT GTT CTT ATT CTG CTT TCT GCT GGT CTT GCC ATCGGC CAT GCT TAT TGG GAA GCA CAG GTG GGC AAT TCC TCT TGG TAC CTC TATGAT GGA GAA GAC GAT ACA CCC TCC TAC CGT GGA TTC CTC ATT TTC TGG GGCTAT ATC ATT GTT CTC AAC ACC ATG GTA CCC ATC TCT CTC TAT GTC AGC GTGGAA GTG ATT CGT CTT GGA CAG AGT CAC TTC ATC AAC TGG GAC CTG CAA ATGTAC TAT GCT GAG AAG GAC ACA CCC GCA AAA GCT AGA ACC ACC ACA CTC AATGAA CAG CTC GGG CAG ATC CAT TAT ATC TTC TCT GAT AAG ACG GGG ACA CTCACA CAA AAT ATC ATG ACC TTT AAA AAG TGC TGT ATC AAC GGG CAG ATA TATGGG GAC CAT CGG GAT GCC TCT CAA CAC AAC CAC AAC AAA ATA GAG CAA GTTGAT TTT AGC TGG AAT ACA TAT GCT GAT GGG AAG CTT GCA TTT TAT GAC CACTAT CTT ATT GAG CAA ATC CAG TCA GGG AAA GAG CCA GAA GTA CGA CAG TTCTTC TTC TTG CTC GCA GTT TGC CAC ACA GTC ATG GTG GAT AGG ACT GAT GGTCAG CTC AAC TAC CAG GCA GCC TCT CCC GAT GAA GGT GCC CTG GTA AAC GCTGCC AGG AAC TTT GGC TTT GCC TTC CTC GCC AGG ACC CAG AAC ACC ATC ACCATC AGT GAA CTG GGC ACT GAA AGG ACT TAC AAT GTT CTT GCC ATT TTG GACTTC AAC AGT GAC CGG AAG CGA ATG TCT ATC ATT GTA AGA ACC CCA GAA GGCAAT ATC AAG CTT TAC TGT AAA GGT GCT GAC ACT GTT ATT TAT GAA CGG TTACAT CGA ATG AAT CCT ACT AAG CAA GAA ACA CAG GAT GCC CTG GAT ATC TTTGCA AAT GAA ACT CTT AGA ACC CTA TGC CTT TGC TAC AAG GAA ATT GAA GAAAAA GAA TTT ACA GAA TGG AAT AAA AAG TTT ATG GCT GCC AGT GTG GCC TCCACC AAC CGG GAC GAA GCT CTG GAT AAA GTA TAT GAG GAG ATT GAA AAA GACTTA ATT CTC CTG GGA GCT ACA GCT ATT GAA GAC AAG CTA CAG GAT GGA GTTCCA GAA ACC ATT TCA AAA CTT GCA AAA GCT GAC ATT AAG ATC TGG GTG CTTACT GGA GAC AAA AAG GAA ACT GCT GAA AAT ATA GGA TTT GCT TGT GAA CTTCTG ACT GAA GAC ACC ACC ATC TGC TAT GGG GAG GAT ATT AAT TCT CTT CTTCAT GCA AGG ATG GAA AAC CAG AGG AAT AGA GGT GGC GTC TAC GCA AAG TTTGCA CCT CCT GTG CAG GAA TCT TTT TTT CCA CCC GGT GGA AAC CGT GCC TTAATC ATC ACT GGT TCT TGG TTG AAT GAA ATT CTT CTC GAG AAA AAG ACC AAGAGA AAT AAG ATT CTG AAG CTG AAG TTC CCA AGA ACA GAA GAA GAA AGA CGGATG CGG ACC CAA AGT AAA AGG AGG CTA GAA GCT AAG AAA GAG CAG CGG CAGAAA AAC TTT GTG GAC CTG GCC TGC GAG TGC AGC GCA GTC ATC TGC TGC CGCGTC ACC CCC AAG CAG AAG GCC ATG GTG GTG GAC CTG GTG AAG AGG TAC AAGAAA GCC ATC ACG CTG GCC ATC GGA GAT GGG GCC AAT GAC GTG AAC ATG ATCAAA ACT GCC CAC ATT GGC GTT GGA ATA AGT GGA CAA GAA GGA ATG CAA GCTGTC ATG TCG AGT GAC TAT TCC TTT GCT CAG TTC CGA TAT CTG CAG AGG CTACTG CTG GTG CAT GGC CGA TGG TCT TAC ATA AGG ATG TGC AAG TTC CTA CGATAC TTC TTT TAC AAA AAC TTT GCC TTT ACT TTG GTT CAT TTC TGG TAC TCCTTC TTC AAT GGC TAC TCT GCG CAG ACT GCA TAC GAG GAT TGG TTC ATC ACCCTC TAC AAC GTG CTG TAC ACC AGC CTG CCC GTG CTC CTC ATG GGG CTG CTCGAC CAG GAT GTG AGT GAC AAA CTG AGC CTC CGA TTC CCT GGG TTA TAC ATAGTG GGA CAA AGA GAC TTA CTA TTC AAC TAT AAG AGA TTC TTT GTA AGC TTGTTG CAT GGG GTC CTA ACA TCG ATG ATC CTC TTC TTC ATA CCT CTT GGA GCTTAT CTG CAA ACC GTA GGG CAG GAT GGA GAG GCA CCT TCC GAC TAC CAG TCTTTT GCC GTC ACC ATT GCC TCT GCT CTT GTA ATA ACA GTC AAT TTC CAG ATTGGC TTG GAT ACT TCT TAT TGG ACT TTT GTG AAT GCT TTT TCA ATT TTT GGAAGC ATT GCA CTT TAT TTT GGC ATC ATG TTT GAC TTT CAT AGT GCT GGA ATACAT GTT CTC TTT CCA TCT GCA TTT CAA TTT ACA GGC ACA GCT TCA AAC GCTCTG AGA CAG CCA TAC ATT TGG TTA ACT ATC ATC CTG GCT GTT GCT GTG TGCTTA CTA CCC GTC GTT GCC ATT CGA TTC CTG TCA ATG ACC ATC TGG CCA TCAGAA AGT GAT AAG ATC CAG AAG CAT CGC AAG CGG TTG AAG GCG GAG GAG CAGTGG CAG CGA CGG CAG CAG GTG TTC CGC CGG GGC GTG TCA ACG CGG CGC TCGGCC TAC GCC TTC TCG CAC CAG CGG GGC TAC GCG GAC CTC ATC TCC TCC GGGCGC AGC ATC CGC AAG AAG CGC TCG CCG CTT GAT GCC ATC GTG GCG GAT GGCACC GCG GAG TAC AGG CGC ACC GGG GAC AGC TGA

TABLE 2 Exemplary ATP8B1 Mutations Amino acid position 3 (e.g., T3K)²⁷Amino acid position 23 (e.g., P23L)⁵Amino acid position 45 (e.g., N45T)^(5,8,9)Amino acid position 46 (e.g., R46X)^(A,25)Amino acid position 62 (e.g., C62R)²⁸Amino acid position 63 (e.g., T63T)⁴¹Amino acid position 70 (e.g., D70N)^(1,6)Amino acid position 71 (e.g., R71H)⁴³Amino acid position 78 (e.g., H78Q)¹⁹Amino acid position 82 (e.g., T82T)⁴¹Amino acid position 92 (e.g., Y92Y)⁴¹Amino acid position 93 (e.g., A93A)⁶Amino acid position 96 (e.g., A96G)²⁷Amino acid position 114 (e.g., E114Q)⁸Amino acid position 127 (e.g., L127P⁶, L127V³⁶)Amino acid position 177 (e.g., T177T)⁶Amino acid position 179 (e.g., E179X)²⁹ Δ Amino acid positions 185-282⁴⁴Amino acid position 197 (e.g., G197Lfs*10)²²Amino acid position 201 (e.g., R201S²⁷, R201H³⁵)Amino acid position 203 (e.g., K203E^(5,8), K203⁹, K203fs²⁵)Amino acid position 205 (e.g., N205fs⁶, N205Kfs*2³⁵)Amino acid position 209 (e.g., P209T)⁴Amino acid position 217 (e.g., S217N)⁴³Amino acid position 232 (e.g., D232D)³⁰Amino acid position 233 (e.g., G233R)³⁸Amino acid position 243 (e.g., L243fs*28)³³Amino acid position 265 (e.g., C265R)²⁵Amino acid position 271 (e.g., R271X¹³, R271R³⁰)Amino acid position 288 (e.g., L288S)⁶Amino acid position 294 (e.g., L294S)⁴³Amino acid position 296 (e.g., R296C)¹¹Amino acid position 305 (e.g., F305I)²⁸Amino acid position 306 (e.g., C306R)²³Amino acid position 307 (e.g., H307L)³⁵Amino acid position 308 (e.g., G308V¹, G308D⁶, G308S³⁵)Amino acid position 314 (e.g., G314S)¹³Amino acid position 320 (e.g., M320Vfs*13)¹¹Amino acid position 337 (e.g., M337R)¹⁸Amino acid position 338 (e.g., N338K)¹⁸Amino acid position 340 (e.g., M340V)¹⁸Amino acid position 344 (e.g., I344F)^(6,20)Amino acid position 349 (e.g., I349T)⁴¹Amino acid position 358 (e.g., G358R)²⁸Amino acid position 367 (e.g., G367G)⁴¹Amino acid position 368 (e.g., N368D)⁴¹Amino acid position 393 (e.g., I393V)²⁷Amino acid position 403 (e.g., S403Y)⁶Amino acid position 407 (e.g., S407N)⁴⁰Amino acid position 412 (e.g., R412P)⁶Amino acid position 415 (e.g., Q415R)²⁷Amino acid position 422 (e.g., D422H)³⁵Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)^(4,11)Amino acid position 453 (e.g., S453Y)⁶Amino acid position 454 (e.g., D454G)⁶Amino acid position 455 (e.g., K455N)⁴³Amino acid position 456 (e.g., T456M^(3,6), T456K³⁵)Amino acid position 457 (e.g., G457G⁶, G457fs*6³³)Amino acid position 469 (e.g., C469G)⁴¹Amino acid position 478 (e.g., H478H)⁴¹Amino acid position 500 (e.g., Y500H)⁶Amino acid position 525 (e.g., R525X)⁴ Δ Amino acid position 529⁶Amino acid position 535 (e.g., H535L⁶, H535N⁴¹)Amino acid position 553 (e.g., P553P)⁴³Amino acid position 554 (e.g., D554N^(1,6), D554A³⁵)Δ Amino acid positions 556-628⁴⁴ A Amino acid positions 559-563³⁵Amino acid position 570 (e.g., L570L)⁴¹Amino acid position 577 (e.g., I577V)¹⁹Amino acid position 581 (e.g., E581K)³⁵Amino acid positions 554 and 581 (e.g., D554A + E581K)³⁵Amino acid position 585 (e.g., E585X)²¹Amino acid position 600 (e.g., R600W^(2,4), R600Q⁶)Amino acid position 602 (e.g., R602X)^(3,6)Amino acid position 628 (e.g., R628W)⁶Amino acid position 631 (e.g., R631Q)²⁸ Δ Amino acid positions 645-699⁴Amino acid position 661 (e.g., I661T)^(1,4,6)Amino acid position 665 (e.g., E665X)^(4,6)Amino acid position 672 (e.g., K672fs⁶, K672Vfs*1³⁵)Amino acid position 674 (e.g., M674T)¹⁹Amino acid positions 78 and 674 (e.g., H78Q/M674T)¹⁹Amino acid position 684 (e.g., D684D)⁴¹Amino acid position 688 (e.g., D688G)⁶Amino acid position 694 (e.g., I694T⁶, I694N¹⁷)Amino acid position 695 (e.g., E695K)²⁷Amino acid position 709 (e.g., K709fs⁶, K709Qfs*41¹³)Amino acid position 717 (e.g., T717N)⁴Amino acid position 733 (e.g., G733R)⁶Amino acid position 757 (e.g., Y757X)⁴Amino acid position 749 (e.g., L749P)²¹Amino acid position 792 (e.g., P792fs)⁶ Δ Amino acid position 795-797⁶Amino acid position 809 (e.g., I809L)²⁷Amino acid position 814 (e.g., K814N)²⁸Amino acid position 833 (e.g., R833Q²⁷, R833W⁴¹)Amino acid position 835 (e.g., K835Rfs*36)³⁵Amino acid position 845 (e.g., K845fs)²⁵Amino acid position 849 (e.g., R849Q)²⁴Amino acid position 853 (e.g., F853S, F853fs)⁶Amino acid position 867 (e.g., R867C¹,R867fs⁶, R867H²³)Amino acid position 885 (e.g., K885T)⁴¹Amino acid position 888 (e.g., T888T)⁴¹Amino acid position 892 (e.g., G892R)⁶Amino acid position 912 (e.g., G912R)³⁵Amino acid position 921 (e.g., S921S)⁴¹Amino acid position 924 (e.g., Y924C)²⁸Amino acid position 930 (e.g., R930X⁶, R930Q²⁸)Amino acid position 941 (e.g., R941X)³⁵Amino acid position 946 (e.g., R946T)⁴¹Amino acid position 952 (e.g., R952Q^(5,9,15), R952X⁶)Amino acid position 958 (e.g., N958fs)⁶Amino acid position 960 (e.g., A960A)⁴¹ Δ A Amino acid position 971⁴³Amino acid position 976 (e.g., A976E⁴¹, A976A⁴³)Amino acid position 981 (e.g., E981K)²⁰Amino acid position 994 (e.g., S994R)⁴Amino acid position 1011 (e.g., L1011fs*18)³³Amino acid position 1012 (e.g., S1012I)¹⁰Amino acid position 1014 (e.g., R1014X)^(6,11)Amino acid position 1015 (e.g., F1015L)²⁷Amino acid position 1023 (e.g., Q1023fs)⁶Amino acid position 1040 (e.g., G1040R)^(1,6)Amino acid position 1044 (e.g., S0144L)³⁴Amino acid position 1047 (e.g., L1047fs)⁶Amino acid position 1050 (e.g., I1050K)³¹Amino acid position 1052 (e.g., L1052R)²⁸Amino acid position 1095 (e.g., W1095X)¹¹Amino acid position 1098 (e.g., V1098X)³⁵Amino acid position 1131 (e.g., Q1131X)⁴⁴Amino acid position 1142 (e.g., A1142Tfs*35)⁴³Amino acid position 1144 (e.g., Y1144Y)⁴³Amino acid position 1150 (e.g., I1150T)⁴¹Amino acid position 1152 (e.g., A1152T)³⁰Amino acid position 1159 (e.g., P1159P)^(25,43)Amino acid position 1164 (e.g., R1164X)⁶Amino acid position 1193 (e.g., R1193fs*39)³³Amino acid position 1197 (e.g., V1197L)⁴¹Amino acid position 1208 (e.g., A1208fs)⁶Amino acid position 1209 (e.g., Y1209Lfs*28)⁴Amino acid position 1211 (e.g., F1211L)²⁷Amino acid position 1219 (e.g., D1219H⁵, D1219G²⁷)Amino acid position 1223 (e.g., S1223S)⁴¹Amino acid position 1233 (e.g., P1233P)⁴¹Amino acid position 1241 (e.g., G1241fs)⁶Amino acid position 1248 (e.g., T1248T)⁴³Splice site mutation IVS3 + 1_ + 3delGTG⁶Splice site mutation IVS3 − 2A > G⁶ IVS6 + 5T > G^(17,25)Splice site mutation IV58 + 1G > T⁶ IVS9 − G > A²⁶ IVS12 + 1G > A²⁵Splice site mutation IVS17 − 1G > A⁶Splice site mutation IVS18 + 2T > C⁶Splice site mutation IVS20 − 4CT > AASplice site mutation IVS21 + 5G > A⁶Splice site mutation IVS23 − 3C > A⁶Splice site mutation IV526 + 2T > A⁶ g.24774-42062del⁴ c.−4C > G⁴¹c.145C > T¹² c.181 − 72G > A⁹ c.182 − 5T > A⁴¹ c.182 − 72G > A⁴¹c.246A > G⁹ c.239G > A³⁹ c.279 + 1_279 + 3delGTG⁴⁶ c.280 − 2A > G⁴⁶c.625_62715delinsACAGTAAT⁴⁶ c.554 + 122C > T⁹ c.555 − 3T > C²⁷c.625 + 5 G > T⁴ Amino acid position 209 (e.g., P209T) and c.625 + 5 G > T⁴ c.628 − 30G > A⁴¹ c.628 − 31C > T⁴¹ c.698 + 1G > T⁴⁶c.698 + 20C > T⁴¹ c.782 − 1G > A⁴⁶ c.782 − 34G > A⁴¹ Δ795-797¹⁴c.782 − 1G > A⁴ c.852A > C²⁷ c.941 − 1G > A⁴⁶ c.1014C > T⁹c.1029 + 35G > A⁹ c.1221 − 8C.G⁴¹ 1226delA¹⁶ c.1429 + 1G > A⁴⁶c.1429 + 2T > G¹³ c.1429 + 49G > A⁴¹ c.1430 − 42A > G⁴¹ c.1493T > C¹²c.1587_1589delCTT⁴⁶ c.1630 + 2T > G²⁷ c.1631 − 10T > A⁴¹c.1637 − 37T > C⁴¹ 1660 G > A¹⁴ 1798 C > T¹⁴ 1799 G > A¹⁴c.1819 − 39_41delAA⁹ c.1819 + 1G > A³¹ c.1820 − 27G > A⁴¹c.1918 + 8C > T²⁷ c.1933 − 1G > AK⁴⁶ c.2097 + 2T > C³²c.2097 + 60T > G⁴¹ c.2097 + 89T > C⁴¹ c.2097 + 97T > G⁴¹c.2210 − 114T > C⁹ 2210delA¹⁶ c.2210 − 45_50dupATAAAA⁹ c.2285 + 29C.T⁴¹c.2285 + 32A > G⁴¹ c.2286 − 4_2286 − 3delinsAA⁴⁶ c.2418 + 5G > A⁴⁶c.2707 + 3G > C²⁷ c.2707 + 9T > G⁴¹ c.2707 + 43A > G⁴¹c.2709 − 59T > C⁴¹ c.2931 + 9A > G⁴¹ c.2931 + 59T > A⁴¹c.2932 − 3C > A⁴⁶ c.2932 + 59T > A⁹ c.2937A > C²⁷ c.3016 − 9C > A³¹c.3033-3034del¹⁹ 3122deITCCTA/ insACATCGATGTTGATGTTAGG⁴⁵ 3318 G > A¹⁴c.3400 + 2T > A⁴⁶ c.3401 − 175C > T⁹ c.3401 − 167C > T⁹c.3401 − 108C > T⁹ c.3531 + 8G > T^(9,15) c.3532 − 15C > T⁹ Δ Phe ex 15⁴Ex1_Ex13del⁶ Ex2_Ex6del³³ Ex12_Ex14del²⁷ Skipped Exon 24⁴⁵del5′UTR-ex18¹¹ c.*11C > T⁴¹ c.*1101 + 366G > A⁷ g.92918del565³¹GC preceding exon 16 (e.g., resulting in a 4 bp deletion)⁴²Frameshift from the 5′ end of exon 16⁴² 5′ 1.4 kb deletion⁴⁶

TABLE 3 Selected ATP8B1 Mutations Associated with PFIC-1 Amino acidposition 23 (e.g., P23L)⁵ Amino acid position 78 (e.g., H78Q)¹⁹ Aminoacid position 93 (e.g., A93A)⁶ Amino acid position 96 (e.g., A96G)²⁷Amino acid position 127 (e.g., L127P)⁶ Amino acid position 197 (e.g.,G197Lfs*10)²² Amino acid position 205 (e.g., N205fs)⁶ Amino acidposition 209 (e.g., P209T)⁴ Amino acid position 233 (e.g., G233R)³⁸Amino acid position 243 (e.g., L243fs*28)³³ Amino acid position 288(e.g., L288S)⁶ Amino acid position 296 (e.g., R296C)¹¹ Amino acidposition 308 (e.g., G308V^(1,6)) Amino acid position 320 (e.g.,M320Vfs*13)¹¹ Amino acid position 403 (e.g., S403Y)⁶ Amino acid position407 (e.g., S407N)⁴⁰ Amino acid position 412 (e.g., R412P)⁶ Amino acidposition 415 (e.g., Q415R)²⁷ Amino acid position 429 (e.g., E429A)⁶Amino acid position 446 (e.g., G446R)⁴ Amino acid position 456 (e.g.,T456M)^(3,6) Amino acid position 457 (e.g., G457G⁶, G457fs*6³³) Aminoacid position 500 (e.g., Y500H)⁶ Amino acid position 525 (e.g., R525X)⁴Δ Amino acid position 529⁶ Amino acid position 535 (e.g., H535L)⁶ Aminoacid position 554 (e.g., D554N)^(1,6) Amino acid position 577 (e.g.,I577V)¹⁹ Amino acid position 585 (e.g., E585X)²¹ Amino acid position 600(e.g., R600W)⁴ Amino acid position 602 (e.g., R602X)^(3,6) Amino acidposition 661 (e.g., I661T)^(4,6) Amino acid position 665 (e.g.,E665X)^(4,6) Δ Amino acid positions 645-699⁴ Amino acid position 672(e.g., K672fs)⁶ Amino acid position 674 (e.g., M674T)¹⁹ Amino acidpositions 78 and 674 (e.g., H78Q/M674T)¹⁹ Amino acid position 688 (e.g.,D688G)⁶ Amino acid position 694 (e.g., I694N)¹⁷ Amino acid position 695(e.g., E695K)²⁷ Amino acid position 709 (e.g., K709fs)⁶ Amino acidposition 717 (e.g., T717N)⁴ Amino acid position 733 (e.g., G733R)⁶ Aminoacid position 749 (e.g., L749P)²¹ Amino acid position 757 (e.g., Y757X)⁴Amino acid position 792 (e.g., P792fs)⁶ Amino acid position 809 (e.g.,I809L)²⁷ Amino acid position 853 (e.g., F853S, F853fs)⁶ Amino acidposition 867 (e.g., R867fs)⁶ Amino acid position 892 (e.g., G892R)⁶Amino acid position 930 (e.g., R930X⁶, R952Q¹⁵) Amino acid position 952(e.g., R952X)⁶ Amino acid position 958 (e.g., N958fs)⁶ Amino acidposition 981 (e.g., E981K)²⁰ Amino acid position 994 (e.g., S994R)⁴Amino acid position 1014 (e.g., R1014X)^(6,11) Amino acid position 1015(e.g., F1015L)²⁷ Amino acid position 1023 (e.g., Q1023fs)⁶ Amino acidposition 1040 (e.g., G1040R)^(1,6) Amino acid position 1047 (e.g.,L1047fs)⁶ Amino acid position 1095 (e.g., W1095X)¹¹ Amino acid position1208 (e.g., A1208fs)⁶ Amino acid position 1209 (e.g., Y1209Lfs*28)⁴Amino acid position 1211 (e.g., F1211L)²⁷ Amino acid position 1219(e.g., D1219H⁵, D1219G²⁷) Splice site mutation IVS3 + 1_+ 3delGTG⁶Splice site mutation IVS3 − 2A > G⁶ IVS6 + 5T > G¹⁷ Splice site mutationIVS8 + 1G > T⁶ IVS9 − G > A²⁶ Splice site mutation IVS17 − 1G > A⁶Splice site mutation IVS18 + 2T > C⁶ Splice site mutation IVS21 + 5G >A⁶ g.24774 − 42062del⁴ c.145C > T¹² c.239G > A³⁹ c.625 + 5 G > T⁴ Aminoacid position 209 (e.g., P209T) and c.625 + 5 G > T⁴ c.782 − 1G > A⁴c.1493T > C¹² c.1630 + 2T > G²⁷ 1660 G > A¹⁴ c.2707 + 3G > C²⁷ c.2097 +2T > C³² c.3033 − 3034del¹⁹ 3318 G > A¹⁴ c.3158 + 8G > T¹⁵ Δ Phe ex 15⁴Ex1_Ex13del⁶ Ex2_Ex6del³³ Ex12_Ex14del²⁷ del5′UTR-ex18¹¹ c.*1101 +366G > A⁷ GC preceding exon 16 (e.g., resulting in a 4 bp deletion)⁴²Frameshift from the 5′ end of exon 16⁴² ^(A) A mutation to ‘X’ denotesan early stop codon

REFERENCES FOR TABLES 2 AND 3

-   ¹ Folmer et al., Hepatology. 2009, vol. 50(5), p. 1597-1605.-   ² Hsu et al., Hepatol Res. 2009, vol. 39(6), p. 625-631.-   ³ Alvarez et al., Hum Mol Genet. 2004, vol. 13(20), p. 2451-2460.-   ⁴ Davit-Spraul et al., Hepatology 2010, vol. 51(5), p. 1645-1655.-   s Vitale et al., J Gastroenterol. 2018, vol. 53(8), p. 945-958.-   ⁶ Klomp et al., Hepatology 2004, vol. 40(1), p. 27-38.-   ⁷ Zarenezhad et al., Hepatitis Monthly: 2017, vol. 17(2); e43500.-   ⁸ Dixon et al., Scientific Reports 2017, vol. 7, 11823.-   ⁹ Painter et al., Eur J Hum Genet. 2005, vol. 13(4), p. 435-439.-   ¹⁰ Deng et al., World J Gastroenterol. 2012, vol. 18(44), p.    6504-6509.-   ¹¹ Giovannoni et al., PLoS One. 2015, vol. 10(12): e0145021.-   ¹² Li et al., Hepatology International 2017, vol. 11, No. 1, Supp.    Supplement 1, pp. S180. Abstract Number: OP284.-   ¹³ Togawa et al., Journal of Pediatric Gastroenterology and    Nutrition 2018, vol. 67, Supp. Supplement 1, pp. S363. Abstract    Number: 615.-   ¹⁴ Miloh et al., Gastroenterology 2006, vol. 130, No. 4, Suppl. 2,    pp. A759-A760. Meeting Info.: Digestive Disease Week Meeting/107th    Annual Meeting of the American-Gastroenterological-Association. Los    Angeles, Calif., USA. May 19.-   ¹⁵ Dröge et al., Zeitschrift fur Gastroenterologie 2015, vol. 53,    No. 12. Abstract Number: A3-27. Meeting Info: 32. Jahrestagung der    Deutschen Arbeitsgemeinschaft zum Studium der Leber. Dusseldorf,    Germany. 22 Jan. 2016-23 Jan. 2016-   ¹⁶ Mizuochi et al., Clin Chim Acta. 2012, vol. 413(15-16), p.    1301-1304.-   ¹⁷ Liu et al., Hepatology International 2009, vol. 3, No. 1, p.    184-185. Abstract Number: PE405. Meeting Info: 19th Conference of    the Asian Pacific Association for the Study of the Liver. Hong Kong,    China. 13 Feb. 2009-16 Feb. 2009-   ¹⁸ McKay et al., Version 2. F1000Res. 2013; 2: 32. DOI:    10.12688/f1000research.2-32.v2-   ¹⁹ Hasegawa et al., Orphanet J Rare Dis. 2014, vol. 9:89.-   ²⁰ Stone et al., J Biol Chem. 2012, vol. 287(49), p. 41139-51.-   ²¹ Kang et al., J Pathol Transl Med. 2019 May 16. doi:    10.4132/jptm.2019.05.03. [Epub ahead of print]-   ²² Sharma et al., BMC Gastroenterol. 2018, vol. 18(1), p. 107.-   ²³ Uegaki et al., Intern Med. 2008, vol. 47(7), p. 599-602.-   ²⁴ Goldschmidt et al., Hepatol Res. 2016, vol. 46(4), p. 306-311.-   ²⁵ Liu et al., J Pediatr Gastroenterol Nutr. 2010, vol. 50(2), p.    179-183.-   ²⁶ Jung et al., J Pediatr Gastroenterol Nutr. 2007, vol. 44(4), p.    453-458.-   ²⁷ Bounford. University of Birmingham. Dissertation Abstracts    International, (2016) Vol. 75, No. 1C. Order No.: AA110588329.    ProQuest Dissertations & Theses.-   ²⁸ Stolz et al., Aliment Pharmacol Ther. 2019, vol. 49(9), p.    1195-1204.-   ²⁹ Ivashkin et al., Hepatology International 2016, vol. 10, No. 1,    Supp. SUPPL. 1, pp. S461. Abstract Number: LBO-38. Meeting Info:    25th Annual Conference of the Asian Pacific Association for the    Study of the Liver, APASL 2016. Tokyo, Japan. 20 Feb. 2016-24 Feb.    2016-   ³⁰ Blackmore et al., J Clin Exp Hepatol. 2013, vol. 3(2), p.    159-161.-   ³¹ Matte et al., J Pediatr Gastroenterol Nutr. 2010, vol. 51(4), p.    488-493.-   ³² Squires et al., J Pediatr Gastroenterol Nutr. 2017, vol.    64(3), p. 425-430.-   ³³ Hayshi et al., EBioMedicine. 2018, vol. 27, p. 187-199.-   ³⁴ Nagasaka et al., J Pediatr Gastroenterol Nutr. 2007, vol.    45(1), p. 96-105.-   ³⁵ Wang et al., PLoS One. 2016; vol. 11(4): e0153114.-   ³⁶ Narchi et al., Saudi J Gastroenterol. 2017, vol. 23(5), p.    303-305.-   ³⁷ Alashkar et al., Blood 2015, vol. 126, No. 23. Meeting Info.:    57th Annual Meeting of the American-Society-of-Hematology. Orlando,    Fla., USA. Dec. 5-8, 2015. Amer Soc Hematol.-   ³⁸ Ferreira et al., Pediatric Transplantation 2013, vol. 17, Supp.    SUPPL. 1, pp. 99. Abstract Number: 239. Meeting Info: IPTA 7th    Congress on Pediatric Transplantation. Warsaw, Poland. 13 Jul.    2013-16 Jul. 2013.-   ³⁹ Pauli-Magnus et al., J Hepatol. 2005, vol. 43(2), p. 342-357.-   ⁴⁰ Jericho et al., Journal of Pediatric Gastroenterology and    Nutrition 2015, vol. 60(3), p. 368-374.-   ⁴¹ van der Woerd et al., PLoS One. 2013, vol. 8(11): e80553.-   ⁴² Copeland et al., J Gastroenterol Hepatol. 2013, vol. 28(3), p.    560-564.-   ⁴³ Dröge et al., J Hepatol. 2017, vol. 67(6), p. 1253-1264.-   ⁴⁴ Chen et al., Journal of Pediatrics 2002, vol. 140(1), p. 119-124.-   ⁴⁵ Jirsa et al., Hepatol Res. 2004, vol. 30(1), p. 1-3.-   ⁴⁶ van der Woerd et al., Hepatology 2015, vol. 61(4), p. 1382-1391.

In some embodiments, the mutation in ATP8B1 is selected from L127P,G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, andG1040R.

Canonical Protein Sequence of ABCB11 - Uniprot ID O95342 (SEQ ID NO: 3)MSDSVILRSI KKFGEENDGF ESDKSYNNDK KSRLQDEKKG DGVRVGFFQL FRFSSSTDIWLMFVGSLCAF LHGIAQPGVL LIFGTMTDVF IDYDVELQEL QIPGKACVNN TIVWTNSSLNQNMTNGTRCG LLNIESEMIK FASYYAGTAV AVLITGYIQI CFWVIAAARQ IQKMRKFYFRRIMRMEIGWF DCNSVGELNT RFSDDINKIN DAIADQMALF IQRMTSTTCG FLLGFFRGWKLTLVIISVSP LIGTGAATIG LSVSKFTDYE LKAYAKAGVV ADEVISSMRT VAAFGGEKREVERYEKNLVF AQRWGIRKGI VMGFFTGFVW CLIFLCYALA FWYGSTLVLD EGEYTPGTLVQIFLSVIVGA LNLGNASPCL EAFATGRAAA TSIFETIDRK PIIDCMSEDG YKLDRIKGEIEFHNVTFHYP SRPEVKILND LNMVIKPGEM TALVGPSGAG KSTALQLIQR FYDPCEGMVTVDGHDIRSLN IQWLRDQIGI VEQEPVLFST TIAENIRYGR EDATMEDIVQ AAKEANAYNFIMDLPQQFDT LVGEGGGQMS GGQKQRVAIA RALIRNPKIL LLDMATSALD NESEAMVQEVLSKIQHGHTI ISVAHRLSTV RAADTIIGFE HGTAVERGTH EELLERKGVY FTLVTLQSQGNQALNEEDIK DATEDDMLAR TFSRGSYQDS LRASIRQRSK SQLSYLVHEP PLAVVDHKSTYEEDRKDKDI PVQEEVEPAP VRRILKFSAP EWPYMLVGSV GAAVNGTVTP LYAFLFSQILGTFSIPDKEE QRSQINGVCL LFVAMGCVSL FTQFLQGYAF AKSGELLTKR LRKFGFRAMLGQDIAWFDDL RNSPGALTTR LATDASQVQG AAGSQIGMIV NSFINVIVAM IIAFSFSWKLSLVILCFFPF LALSGATQTR MLTGFASRDK QALEMVGQIT NEALSNIRTV AGTGKERRFIEALETELEKP FKTAIQKANI YGFCFAFAQC IMFIANSASY RYGGYLISNE GLHFSYVFRVISAVVLSATA LGRAFSYTPS YAKAKISAAR FFQLLDRQPP ISVYNTAGEK WDNFQGKIDFVDCKFTYPSR PDSQVLNGLS VSISPGQTLA FVGSSGCGKS TSIQLLERFY DPDQGKVMIDGHDSKKVNVQ FLRSNIGIVS QEPVLFACSI MDNIKYGDNT KEIPMERVIA AAKQAQLHDFVMSLPEKYET NVGSQGSQLS RGEKQRIAIA RAIVRDPKIL LLDEATSALD TESEKTVQVALDKAREGRTC IVIAHRLSTI QNADIIAVMA QGVVIEKGTH EELMAQKGAY YKLVTTGSPI SCanonical DNA Sequence of ABCB11 (SEQ ID NO: 4)ATG TCT GAC TCA GTA ATT CTT CGA AGT ATA AAG AAA TTT GGA GAG GAG AATGAT GGT TTT GAG TCA GAT AAA TCA TAT AAT AAT GAT AAG AAA TCA AGG TTACAA GAT GAG AAG AAA GGT GAT GGC GTT AGA GTT GGC TTC TTT CAA TTG TTTCGG TTT TCT TCA TCA ACT GAC ATT TGG CTG ATG TTT GTG GGA AGT TTG TGTGCA TTT CTC CAT GGA ATA GCC CAG CCA GGC GTG CTA CTC ATT TTT GGC ACAATG ACA GAT GTT TTT ATT GAC TAC GAC GTT GAG TTA CAA GAA CTC CAG ATTCCA GGA AAA GCA TGT GTG AAT AAG ACC ATT GTA TGG ACT AAG AGT TCC CTCAAC CAG AAC ATG ACA AAT GGA ACA CGT TGT GGG TTG CTG AAC ATC GAG AGCGAA ATG ATC AAA TTT GCC AGT TAC TAT GCT GGA ATT GCT GTC GCA GTA CTTATC ACA GGA TAT ATT CAA ATA TGC TTT TGG GTC ATT GCC GCA GCT CGT CAGATA CAG AAA ATG AGA AAA TTT TAC TTT AGG AGA ATA ATG AGA ATG GAA ATAGGG TGG TTT GAC TGC AAT TCA GTG GGG GAG CTG AAT ACA AGA TTC TCT GATGAT ATT AAT AAA ATC AAT GAT GCC ATA GCT GAC CAA ATG GCC CTT TTC ATTCAG CGC ATG ACC TCG ACC ATC TGT GGT TTC CTG TTG GGA TTT TTC AGG GGTTGG AAA CTG ACC TTG GTT ATT ATT TCT GTC AGC CCT CTC ATT GGG ATT GGAGCA GCC ACC ATT GGT CTG AGT GTG TCC AAG TTT ACG GAC TAT GAG CTG AAGGCC TAT GCC AAA GCA GGG GTG GTG GCT GAT GAA GTC ATT TCA TCA ATG AGAACA GTG GCT GCT TTT GGT GGT GAG AAA AGA GAG GTT GAA AGG TAT GAG AAAAAT CTT GTG TTC GCC CAG CGT TGG GGA ATT AGA AAA GGA ATA GTG ATG GGATTC TTT ACT GGA TTC GTG TGG TGT CTC ATC TTT TTG TGT TAT GCA CTG GCCTTC TGG TAC GGC TCC ACA CTT GTC CTG GAT GAA GGA GAA TAT ACA CCA GGAACC CTT GTC CAG ATT TTC CTC AGT GTC ATA GTA GGA GCT TTA AAT CTT GGCAAT GCC TCT CCT TGT TTG GAA GCC TTT GCA ACT GGA CGT GCA GCA GCC ACCAGC ATT TTT GAG ACA ATA GAC AGG AAA CCC ATC ATT GAC TGC ATG TCA GAAGAT GGT TAC AAC TTG GAT CGA ATC AAG GGT GAA ATT GAA TTC CAT AAT GTGACC TTC CAT TAT CCT TCC AGA CCA GAG GTG AAG ATT CTA AAT GAC CTC AACATG GTC ATT AAA CCA GGG GAA ATG ACA GCT CTG GTA GGA CCC AGT GGA GCTGGA AAA AGT ACA GCA CTG CAA CTC ATT CAG CGA TTC TAT GAC CCC TGT GAAGGA ATG GTG ACC GTG GAT GGC CAT GAC ATT CGC TCT CTT AAC ATT CAG TGGCTT AGA GAT CAG ATT GGG ATA GTG GAG CAA GAG CCA GTT CTG TTC TCT ACCACC ATT GCA GAA AAT ATT CGC TAT GGC AGA GAA GAT GCA ACA ATG GAA GACATA GTC CAA GCT GCC AAG GAG GCC AAT GCC TAC AAC TTC ATC ATG GAC CTGCCA CAG CAA TTT GAC ACC CTT GTT GGA GAA GGA GGA GGC CAG ATG AGT GGTGGC CAG AAA CAA AGG GTA GCT ATC GCC AGA GCC CTC ATC CGA AAT CCC AAGATT CTG CTT TTG GAC ATG GCC ACC TCA GCT CTG GAC AAT GAG AGT GAA GCCATG GTG CAA GAA GTG CTG AGT AAG ATT CAG CAT GGG CAC ACA ATC ATT TCAGTT GCT CAT CGC TTG TCT ACG GTC AGA GCT GCA GAT ACC ATC ATT GGT TTTGAA CAT GGC ACT GCA GTG GAA AGA GGG ACC CAT GAA GAA TTA CTG GAA AGGAAA GGT GTT TAC TTC ACT CTA GTG ACT TTG CAA AGC CAG GGA AAT CAA GCTCTT AAT GAA GAG GAC ATA AAG GAT GCA ACT GAA GAT GAC ATG CTT GCG AGGACC TTT AGC AGA GGG AGC TAC CAG GAT AGT TTA AGG GCT TCC ATC CGG CAACGC TCC AAG TCT CAG CTT TCT TAC CTG GTG CAC GAA CCT CCA TTA GCT GTTGTA GAT CAT AAG TCT ACC TAT GAA GAA GAT AGA AAG GAC AAG GAC ATT CCTGTG CAG GAA GAA GTT GAA CCT GCC CCA GTT AGG AGG ATT CTG AAA TTC AGTGCT CCA GAA TGG CCC TAC ATG CTG GTA GGG TCT GTG GGT GCA GCT GTG AACGGG ACA GTC ACA CCC TTG TAT GCC TTT TTA TTC AGC CAG ATT CTT GGG ACTTTT TCA ATT CCT GAT AAA GAG GAA CAA AGG TCA CAG ATC AAT GGT GTG TGCCTA CTT TTT GTA GCA ATG GGC TGT GTA TCT CTT TTC ACC CAA TTT CTA CAGGGA TAT GCC TTT GCT AAA TCT GGG GAG CTC CTA ACA AAA AGG CTA CGT AAATTT GGT TTC AGG GCA ATG CTG GGG CAA GAT ATT GCC TGG TTT GAT GAC CTCAGA AAT AGC CCT GGA GCA TTG ACA ACA AGA CTT GCT ACA GAT GCT TCC CAAGTT CAA GGG GCT GCC GGC TCT CAG ATC GGG ATG ATA GTC AAT TCC TTC ACTAAC GTC ACT GTG GCC ATG ATC ATT GCC TTC TCC TTT AGC TGG AAG CTG AGCCTG GTC ATC TTG TGC TTC TTC CCC TTC TTG GCT TTA TCA GGA GCC ACA CAGACC AGG ATG TTG ACA GGA TTT GCC TCT CGA GAT AAG CAG GCC CTG GAG ATGGTG GGA CAG ATT ACA AAT GAA GCC CTC AGT AAC ATC CGC ACT GTT GCT GGAATT GGA AAG GAG AGG CGG TTC ATT GAA GCA CTT GAG ACT GAG CTG GAG AAGCCC TTC AAG ACA GCC ATT CAG AAA GCC AAT ATT TAC GGA TTC TGC TTT GCCTTT GCC CAG TGC ATC ATG TTT ATT GCG AAT TCT GCT TCC TAC AGA TAT GGAGGT TAC TTA ATC TCC AAT GAG GGG CTC CAT TTC AGC TAT GTG TTC AGG GTGATC TCT GCA GTT GTA CTG AGT GCA ACA GCT CTT GGA AGA GCC TTC TCT TACACC CCA AGT TAT GCA AAA GCT AAA ATA TCA GCT GCA CGC TTT TTT CAA CTGCTG GAC CGA CAA CCC CCA ATC AGT GTA TAC AAT ACT GCA GGT GAA AAA TGGGAC AAC TTC CAG GGG AAG ATT GAT TTT GTT GAT TGT AAA TTT ACA TAT CCTTCT CGA CCT GAC TCG CAA GTT CTG AAT GGT CTC TCA GTG TCG ATT AGT CCAGGG CAG ACA CTG GCG TTT GTT GGG AGC AGT GGA TGT GGC AAA AGC ACT AGCATT CAG CTG TTG GAA CGT TTC TAT GAT CCT GAT CAA GGG AAG GTG ATG ATAGAT GGT CAT GAC AGC AAA AAA GTA AAT GTC CAG TTC CTC CGC TCA AAC ATTGGA ATT GTT TCC CAG GAA CCA GTG TTG TTT GCC TGT AGC ATA ATG GAC AATATC AAG TAT GGA GAC AAC ACC AAA GAA ATT CCC ATG GAA AGA GTC ATA GCAGCT GCA AAA CAG GCT CAG CTG CAT GAT TTT GTC ATG TCA CTC CCA GAG AAATAT GAA ACT AAC GTT GGG TCC CAG GGG TCT CAA CTC TCT AGA GGG GAG AAACAA CGC ATT GCT ATT GCT CGG GCC ATT GTA CGA GAT CCT AAA ATC TTG CTACTA GAT GAA GCC ACT TCT GCC TTA GAC ACA GAA AGT GAA AAG ACG GTG CAGGTT GCT CTA GAC AAA GCC AGA GAG GGT CGG ACC TGC ATT GTC ATT GCC CATCGC TTG TCC ACC ATC CAG AAC GCG GAT ATC ATT GCT GTC ATG GCA CAG GGGGTG GTG ATT GAA AAG GGG ACC CAT GAA GAA CTG ATG GCC CAA AAA GGA GCCTAC TAC AAA CTA GTC ACC ACT GGA TCC CCC ATC AGT TGA

TABLE 4 Exemplary ABCB11 Mutations Amino acid position 1 (e.g., M1V)⁹Amino acid position 4 (e.g., S4X)^(A,64) Amino acid position 8 (e.g.,R8X)⁸⁸ Amino acid position 19 (e.g., G19R)⁵⁶ Amino acid position 24(e.g., K24X)³⁵ Amino acid position 25 (e.g., S25X)^(5,14) Amino acidposition 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 36 (e.g., D36D)²⁷Amino acid position 38 (e.g., K38Rfs*24)⁷³ Amino acid position 43 (e.g.,V43I)⁵⁷ Amino acid position 49 (e.g., Q49X)⁷³ Amino acid position 50(e.g., L50S, L50W)⁵⁷ Amino acid position 52 (e.g., R52W²⁶, R52R²⁸) Aminoacid position 56 (e.g., S56L)⁵⁸ Amino acid position 58 (e.g., D58N)⁶²Amino acid position 62 (e.g., M62K)⁹ Amino acid position 66 (e.g.,S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Amino acid position 50(e.g., L50S)^(5,7) Amino acid position 71 (e.g., L71H)⁷³ Amino acidposition 74 (e.g., I74R)⁷¹ Amino acid position 77 (e.g., P77A)⁷³ Aminoacid position 87 (e.g., T87R)⁶⁷ Amino acid position 90 (e.g.,F90F)^(7,27) Amino acid position 93 (e.g., Y93S¹³, Y93X⁸⁸) Amino acidposition 96 (e.g., E96X)⁸⁸ Amino acid position 97 (e.g., L97X)³⁹ Aminoacid position 101 (e.g., Q101Dfs*8)⁹ Amino acid position 107 (e.g.,C107R)³⁶ Amino acid position 112 (e.g., I112T)⁹ Amino acid position 114(e.g., W114R)^(2,9) Amino acid position 123 (e.g. M123T)⁶⁷ Amino acidposition 127 (e.g., T127Hfs*6)⁵ Amino acid position 129 (e.g., C129Y)²⁵Amino acid position 130 (e.g., G130G)⁷⁷ Amino acid position 134 (e.g.,I134I)²⁸ Amino acid position 135 (e.g., E135K^(7,13), E135L¹⁷) Aminoacid position 137 (e.g., E137K)⁷ Amino acid position 157 (e.g., Y157C)⁵Amino acid position 161 (e.g., C161X)³⁹ Amino acid position 164 (e.g.,V164Gfs*7³⁰, V164I⁸⁵) Amino acid position 167 (e.g., A167S⁴, A167V⁷,A167T^(9,17)) Amino acid position 181 (e.g., R181I)³⁵ Amino acidposition 182 (e.g., I182K)⁹ Amino acid position 183 (e.g., M183V⁸,M183T⁹) Amino acid position 185 (e.g., M185I)⁷³ Amino acid position 186(e.g., E186G)^(2,7,22) Amino acid position 188 (e.g., G188W)⁷³ Aminoacid position 194 (e.g., S194P)⁷ Amino acid position 198 (e.g., L198P)⁷Amino acid position 199 (e.g., N199Ifs*15X)⁸⁸ Amino acid position 206(e.g., 1206V)²⁸ Amino acid position 212 (e.g., A212T)⁷³ Amino acidposition 217 (e.g., M217R)⁸⁸ Amino acid position 225 (e.g., T225P)⁵⁷Amino acid position 226 (e.g., S226L)⁹ Amino acid position 232 (e.g.,L232Cfs*9)⁹ Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position238 (e.g., G238V)^(2,7) Amino acid position 242 (e.g., T242I)^(5,7)Amino acid position 245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256(e.g., A256G)⁹ Amino acid position 260 (e.g., G260D)⁷ Amino acidposition 269 (e.g., Y269Y)²⁷ Amino acid position 277 (e.g., A277E)⁷⁷Amino acid position 283 (e.g., E283D)⁷³ Amino acid positions 212 and 283(e.g., A212T + E283D)⁷³ Amino acid position 284 (e.g., V284L^(7,39),V284A⁷, V284D²³) Amino acid position 297 (e.g., E297G^(1,2,5,7), E297K⁷)Amino acid position 299 (e.g., R299K)²⁸ Amino acid position 303 (e.g.,R303K⁸, R303M⁶³ R303fsX321⁸³) Amino acid position 304 (e.g., Y304X)²⁶Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313 (e.g.,R313S)^(5,7) Amino acid position 314 (e.g., W314X)⁵⁷ Amino acid position318 (e.g., K318Rfs*26)²⁹ Amino acid position 319 (e.g., G319G)⁷ Aminoacid position 327 (e.g., G327E)^(5,7) Amino acid position 330 (e.g.,W330X)²⁴ Amino acid position 336 (e.g., C336S)^(2,7) Amino acid position337 (e.g., Y337H)^(21,27) Amino acid position 342 (e.g., W342G)⁵⁰ Aminoacid position 354 (e.g., R354X)⁹ Amino acid position 361 (e.g., Q361X⁵⁷,Q361R⁷⁴) Amino acid position 366 (e.g., V366V²⁸, V366D⁵⁷) Amino acidposition 368 (e.g., V368Rfs*27)⁵ Amino acid position 374 (e.g., G374S)³Amino acid position 380 (e.g., L380Wfs*18)⁵ Amino acid position 382(e.g., A382G)⁸⁸ Δ Amino acid positions 382-388⁵ Δ Amino acid positions383-389⁵⁷ Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390(e.g., A390P)^(5,7) Amino acid position 395 (e.g., E395E)²⁸ Amino acidposition 404 (e.g., D404G)⁹ Amino acid position 410 (e.g., G410D)^(5,7)Amino acid position 413 (e.g., L413W)^(5,7) Amino acid position 415(e.g., R415X)⁴² Amino acid position 416 (e.g., I416I)²⁷ Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 423 (e.g., H423R)¹³Amino acid position 432 (e.g., R432T)^(1,2,7) Amino acid position 436(e.g., K436N)⁴⁰ Amino acid position 440 (e.g., D440E)⁸⁸ Amino acidposition 444 (e.g., V444A)² Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 457 (e.g.,S457Vfs*23)⁸⁸ Amino acid position 461 (e.g., K461E)^(2,7) Amino acidposition 462 (e.g., S462R)⁸⁸ Amino acid position 463 (e.g., T463I)^(5,7)Amino acid position 466 (e.g., Q466K)^(5,7) Amino acid position 470(e.g., R470Q^(5,7), R470X⁹) Amino acid position 471 (e.g., Y472X)⁵ Aminoacid position 472 (e.g., Y472C^(5,27), Y472X¹⁴) Amino acid position 473(e.g., D473Q³⁵, D473V⁸⁸) Amino acid position 475 (e.g., C475X)²⁹ Aminoacid position 481 (e.g., V481E)^(5,7) Amino acid position 482 (e.g.,D482G)^(2,5,7) Amino acid position 484 (e.g., H484Rfs*5)⁹ Amino acidposition 487 (e.g., R487H², R487P⁵) Amino acid position 490 (e.g.,N490D)^(5,7) Amino acid position 493 (e.g., W493X)⁸ Amino acid positon496 (e.g., D496V)⁸⁸ Amino acid position 498 (e.g., I498T)^(2,7) Aminoacid position 499 (e.g., G499E)⁷³ Amino acid position 501 (e.g.,V501G)⁶⁸ Amino acid position 504 (e.g., E504K)⁷⁹ Amino acid position 510(e.g., T510T)⁷ Amino acid position 512 (e.g., I512T)^(5,7) Amino acidposition 515 (e.g., N515T^(5,7), N515D⁶⁴) Amino acid position 516 (e.g.,I516M)¹⁷ Amino acid position 517 (e.g., R517H)^(5,7) Amino acid position520 (e.g., R520X)⁵ Amino acid position 523 (e.g., A523G)¹³ Amino acidposition 528 (e.g., I528Sfs*21⁵, I528X⁹, I528T⁷³) Amino acid position535 (e.g., A535A⁷, A535X⁸⁹) Amino acid position 540 (e.g., F540L)⁴⁶Amino acid position 541 (e.g., I541L^(5,7), I541T^(5,17)) Amino acidposition 546 (e.g., Q546K³⁹, Q546H⁷³) Amino acid position 548 (e.g.,F548Y)^(5,7) Amino acid position 549 (e.g., D549V)⁹ Amino acid position554 (e.g., E554K)²¹ Amino acid position 556 (e.g., G556R)⁶⁷ Amino acidposition 558 (e.g., Q558H)²³ Amino acid position 559 (e.g., M559T)⁵⁷Amino acid position 562 (e.g., G562D^(5,7), G562S⁷³) Amino acid position570 (e.g., A570T^(2,5,7), A570V²⁶) Amino acid position 575 (e.g.,R575X^(2,5), R575Q²¹) Amino acid position 580 (e.g., L580P)⁵⁷ Amino acidposition 586 (e.g., T586I)⁷ Amino acid position 587 (e.g., S587X)⁷³Amino acid position 588 (e.g., A588V^(5,7), A588P⁷³) Amino acid position591 (e.g., N591S)^(2,7) Amino acid position 593 (e.g., S593R)^(2,7)Amino acid position 597 (e.g., V597V⁹, V597L¹³) Amino acid position 603(e.g., K603K)⁵⁵ Amino acid position 609 (e.g., H609Hfs*46)²⁶ Amino acidposition 610 (e.g., I610Gfs*45⁹, I610T⁵⁷)⁹ Amino acid position 615(e.g., H615R)²⁶ Amino acid position 616 (e.g., R616G²⁸, R616H⁷³) Aminoacid position 619 (e.g., T619A)²⁸ Amino acid position 623 (e.g.,A623A)²⁸ Amino acid position 625 (e.g., T625Nfs*5)²⁶ Amino acid position627 (e.g., I627T)⁷ Amino acid position 628 (e.g., G628Wfs*3)⁷⁰ Aminoacid position 636 (e.g., E636G)² Amino acid position 648 (e.g.,G648Vfs*6⁵, G648V⁵⁰) Amino acid position 655 (e.g., T655I)⁷ Amino acidposition 669 (e.g., I669V)²⁶ Amino acid position 676 (e.g., D676Y)¹¹Amino acid position 677 (e.g., M677V)^(7,13) Amino acid position 679(e.g., A679V)⁵⁸ Amino acid position 685 (e.g., G685W)⁶⁰ Amino acidposition 696 (e.g., R696W²⁷, R696Q⁵⁸) Amino acid position 698 (e.g.,R698H^(7,9), R698K⁶¹, R698C⁸⁸) Amino acid position 699 (e.g., S699P)⁹Amino acid position 701 (e.g., S701P)⁵⁸ Amino acid position 702 (e.g.,Q702X)⁸⁹ Amino acid position 709 (e.g., E709K)⁷ Amino acid position 710(e.g., P710P)⁷ Amino acid position 712 (e.g., L712L)²⁸ Amino acidposition 721 (e.g., Y721C)⁸⁸ Amino acid position 729 (e.g., D724N)³⁹Amino acid position 731 (e.g., P731S)²³ Amino acid position 740 (e.g.,P740Qfs*6)⁷³ Amino acid position 758 (e.g., G758R)⁵ Amino acid position766 (e.g., G766R)^(5,24) Amino acid position 772 (e.g., Y772X)⁵ Aminoacid position 804 (e.g., A804A)⁷ Amino acid position 806 (e.g., G806D⁴⁴,G806G⁵⁵) Amino acid position 809 (e.g., S809F)⁸¹ Amino acid position 817(e.g., G817G)⁸⁸ Amino acid position 818 (e.g., Y818F)⁷ Amino acidposition 824 (e.g., G824E)⁴² Amino acid position 825 (e.g., G825G)⁷³Amino acid position 830 (e.g., R830Gfs*28)⁷³ Amino acid position 832(e.g., R832C^(7,26), R832H⁴¹) Amino acid position 842 (e.g., D842G)²Amino acid position 848 (e.g., D848N)⁷³ Amino acid position 855 (e.g.,G855R)¹¹ Amino acid position 859 (e.g., T859R)^(5,7) Amino acid position865 (e.g., A865V)²⁷ Amino acid position 866 (e.g., S866A)⁵⁷ Amino acidposition 868 (e.g., V868D)⁷³ Amino acid position 869 (e.g., Q869P)⁷³Amino acid position 875 (e.g., Q875X)⁷³ Amino acid position 877 (e.g.,G877R)⁵⁶ Amino acid position 879 (e.g., I879R)⁸⁸ Amino acid position 893(e.g., A893V)⁵⁷ Amino acid position 901 (e.g., S901R¹⁷, S901I⁷³) Aminoacid position 903 (e.g., V903G)⁵⁷ Δ Amino acid position 919¹² Amino acidposition 923 (e.g., T923P)^(2,7) Amino acid position 926 (e.g.,A926P)^(2,7) Amino acid position 928 (e.g., R928X¹⁵, R928Q⁴⁰) Amino acidposition 930 (e.g., K930X⁵, K930Efs*79^(5,10), K930Efs*49²⁶) Amino acidposition 931 (e.g., Q931P)²⁷ Amino acid position 945 (e.g., S945N)⁵⁷Amino acid position 948 (e.g., R948C)^(5,7,26) Amino acid position 958(e.g., R958Q)²⁸ Amino acid position 969 (e.g., K969K)⁸⁸ Δ Amino acidpositions 969-972⁵ Amino acid position 973 (e.g., T973I)⁵⁷ Amino acidposition 976 (e.g., Q976R⁵⁸, Q976X⁸⁸) Amino acid position 979 (e.g.,N979D)^(5,7) Amino acid position 981 (e.g., Y981Y)²⁸ Amino acid position982 (e.g., G982R)^(2,5,7) Amino acid positions 444 and 982 (e.g.,V444A + G982R)³⁸ Amino acid position 995 (e.g., A995A)²⁸ Amino acidposition 1001 (e.g., R1001R)⁹ Amino acid position 1003 (e.g., G1003R)²⁴Amino acid position 1004 (e.g., G1004D)^(2,7) Amino acid position 1027(e.g., S1027R)²⁶ Amino acid position 1028 (e.g., A1028A^(7,10,88),A1028E⁸⁸) Amino acid position 1029 (e.g., T1029K)⁵ Amino acid position1032 (e.g., G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Aminoacid position 1044 (e.g., A1044P)⁸⁸ Amino acid position 1050 (e.g.,R1050C)^(2,7,57) Amino acid position 1053 (e.g., Q1053X)⁵⁷ Amino acidposition 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g., R1057X²,R1057Q⁵⁸) Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷,Q1058X⁷³) Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acidposition 1083 (e.g., C1083Y)⁴⁷ Amino acid position 1086 (e.g., T1086T)²⁸Amino acid position 1090 (e.g., R1090X)^(2,5) Amino acid position 1099(e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g., S1100Qfs*38)¹³Amino acid position 1110 (e.g., A1110E)^(5,7) Amino acid position 1112(e.g., V1112F)⁷⁰ Amino acid position 1116 (e.g., G1116R⁷, G1116F^(9,17),G1116E³⁶) Amino acid position 1120 (e.g., S1120N)⁸⁸ Amino acid position1128 (e.g., R1128H^(2,7), R1128C^(5,7,13)) Amino acid position 1131(e.g., D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acidposition 1147 (e.g., V1147X)⁵ Amino acid position 1153 (e.g.,R1153C^(2,5,7), R1153H⁵) Amino acid position 1154 (e.g., S1154P)^(5,7)Amino acid position 1162 (e.g., E1162X)³⁹ Δ Amino acid position 1165⁸⁸Amino acid position 1164 (e.g., V1164Gfs*7) Amino acid position 1173(e.g., N1173D)⁵⁷ Amino acid position 1175 (e.g., K1175T)⁵⁸ Amino acidposition 1186 (e.g., E1186K)⁷ Amino acid position 1192 (e.g.,A1192Efs*50)⁹ Amino acid position 1196 (e.g., Q1196X)⁸⁸ Amino acidposition 1197 (e.g., L1197G)⁷ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1204 (e.g., L1204P)⁸⁸ Amino acid position 1208 (e.g.Y1208C)⁷³ Amino acid position 1210 (e.g., T1210P^(5,7), T1210F⁵⁷) Aminoacid position 1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g.,V1212F)³⁶ Amino acid position 1215 (e.g., Q1215X)⁵ Amino acid position1221 (e.g., R1221K)⁵³ Amino acid position 1223 (e.g., E1223D)⁷ Aminoacid position 1226 (e.g., R1226P)⁷³ Amino acid position 1228 (e.g.,A1228V)⁷ Amino acid position 1231 (e.g., R1231W^(5,7), R1231Q^(5,7))Amino acid position 1232 (e.g., A1232D)¹⁷ Amino acid position 1235(e.g., R1235X)^(5,12) Amino acid position 1242 (e.g., L1242I)^(5,7)Amino acid position 1243 (e.g., D1243G)⁶⁷ Amino acid position 1249(e.g., L1249X)⁷³ Amino acid position 1256 (e.g., T1256fs*1296)⁸³ Aminoacid position 1268 (e.g., R1268Q)^(2,7) Amino acid position 1276 (e.g.,R1276H)³⁰ Amino acid position 1283 (e.g., A1283A²⁸, A1283V⁸⁸) Amino acidposition 1292 (e.g., G1292V)⁷³ Amino acid position 1298 (e.g., G1298R)⁵Amino acid position 1302 (e.g., E1302X)⁵ Amino acid position 1311 (e.g.,Y1311X)⁵⁷ Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Amino acidposition 1321 (e.g., S1321N)⁵⁷ Intron 4 ((+3)A > C)¹ IVS4 − 74A > T⁸⁹Splice site mutation 3′ Intron 5 c.3901G > A⁵ Splice site mutation 5;Intron 7 c.6111G > A⁵ Splice site mutation IVS7 + 1G > A¹⁴ IVS7 + 5G >A⁴⁰ IVS8 + 1G > C⁷⁶ Splice site mutation 5′ Intron 9 c.9081delG⁵ Splicesite mutation 5′ Intron 9 c.9081G > T⁵ Splice site mutation 5′ Intron 9c.9081G > A⁵ Splice site mutation IVS9 + 1G > T¹⁴ Splice site mutation3′ Intron 13 c.143513_1435 − 8del⁵ Splice site mutation IVS13del −13{circumflex over ( )} − 8¹⁴ Splice site mutation 3′ Intron 16c.20128T > G⁵ Splice site mutation IVS16 − 8T > G¹⁴ Splice site mutation5′ Intron 18 c.21781G > T⁵ Splice site mutation 5′ Intron 18 c.21781G >A⁵ Splice site mutation 5′ Intron 18 c.21781G > C⁵ Splice site mutation3′ Intron 18 c.21792A > G⁵ Splice site mutation IVS18 + 1G > A¹⁴ Splicesite mutation 5′ Intron 19 c.2343 + 1G > T⁵ Splice site mutation 5′Intron 19 c.2343 + 2T > C⁵ Splice site mutation IVS19 + 2T > C¹⁴ Splicesite mutation IVS19 + 1G > A²² Splice site mutation 3′ Intron 21c.26112A > T⁵ IVS22 + 3A > G⁸⁹ IVS 23 − 8 G − A³⁶ IVS24 + 5G > A⁵¹Splice site mutation 5′ Intron 24 c.32131delG⁵ IVS35 − 6C > G⁸⁹ Putativesplice mutation 1198 − 1G > C¹⁷ Putative splice mutation 1810 − 3C > G¹⁷Putative splice mutation 2178 + 1G > A¹⁷ Putative splice mutation 2344 −1G > T¹⁷ Putative splice mutation c.2611 − 2A > T³⁹ Putative splicemutation 3213 + 1_3213 + 2delinsA¹⁷ c.−24C > A^(44,78) c.76 13 G > T⁹c.77 − 19T > A⁵² c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰ 174C >T⁵⁴ c.245T > C⁸⁷ c.249_250insT¹⁸ 270T > C⁵⁴ 402C > T⁵⁴ 585G > C⁵⁴c.611 + 1G > A⁷⁰ c.611 + 4A > G³⁶ c.612 − 15_− 6del10 bp⁵⁵ c.625A > C³¹c.627 + 5G > T³¹ c.625A > C/c.627 + 5G > T³¹ 696G > T⁵⁴ c. 784 + 1G >C⁴⁹ 807T > C⁵⁴ c.886C > T³¹ c.890A > G⁵⁹ c.908 + 1G > A⁵⁷ c.908 + 5G >A⁵⁵ c.908delG⁵⁹ c.909 − 15A > G⁶⁶ 957A > G⁵⁴ c.1084 − 2A > G⁵⁷ 1145 1 bpdeletion⁹⁰ 1281C > T^(54,57) c.1309 − 165C > T¹⁹ c.1434 + 174G > A¹⁹c.1434 + 70C > T¹⁹ c.1530C > A⁵⁷ c.1587 − 1589delCTT³¹ c.1621A >C^(33,59) c.1638 + 32T > C⁶⁶ c.1638 + 80C > T⁶⁶ 1671C > T⁵⁴ 1791G > T⁵⁴1939delA¹⁴ c.2075 + 3A > G⁵³ c.2081T > A³¹ c.2093G > A⁶⁵ 2098delA¹⁶c.2138 − 8T > G⁶⁷ 2142A > G⁵⁴ c.2178 + 1G > T^(36,39) c.2179 − 17C > A⁶⁶c.2344 − 157T > G⁶⁶ c.2344 − 17T > C⁶⁶ c.2417G > A⁷⁸ c.2541delG⁸⁷c.2620C > T^(32,33) c.2815 − 8A > G⁵⁵ c.3003A > G³⁷ c.3084A > G^(48,54)c.3213 + 4 A > G^(9,37) c.3213 + 5 G > A⁹ c.3268C > T⁷⁵ 3285A > G⁵⁴c.3382C > T⁷⁵ 3435A > G⁵⁴ c.3491delT⁷² c.3589C > T⁵⁷ c.3765(+1 +5)del5⁴² c.3766 − 34A > G⁶⁶ c.3767 − 3768insC⁶ c.3770delA⁶⁷ c.3826C >T⁷² c.3846C > T⁵⁷ c.3929delG⁶⁷ c.*236A > G⁶⁶ 1145delC⁸ Ex13_Ex17del⁸²

TABLE 5 Selected ABCB11 Mutations Associated with PFIC-2 Amino acidposition 1 (e.g., M1V)⁹ Amino acid position 4 (e.g., S4X)⁶⁴ Amino acidposition 19 (e.g., G19R)⁵⁶ Amino acid position 25 (e.g., S25X)¹⁴ Aminoacid position 26 (e.g., Y26Ifs*7)³⁸ Amino acid position 50 (e.g.,L50S)^(7,57) Amino acid position 52 (e.g., R52W)²⁶ Amino acid position58 (e.g., D58N)⁶² Amino acid position 62 (e.g., M62K)⁹ Amino acidposition 66 (e.g., S66N)¹⁷ Amino acid position 68 (e.g., C68Y)⁴¹ Aminoacid position 93 (e.g., Y93S)¹³ Amino acid position 101 (e.g.,Q101Dfs*8)⁹ Amino acid position 107 (e.g., C107R)³⁶ Amino acid position112 (e.g., I112T)⁹ Amino acid position 114 (e.g., W114R)^(2,9) Aminoacid position 129 (e.g., C129Y)²⁵ Amino acid position 135 (e.g.,E135K¹³, E135L¹⁷) Amino acid position 167 (e.g., A167V⁷, A167T^(9,17))Amino acid position 182 (e.g., I182K)⁹ Amino acid position 183 (e.g.,M183V⁸, M183T⁹) Amino acid position 225 (e.g., T225P)⁵⁷ Amino acidposition 226 (e.g., S226L)⁹ Amino acid position 232 (e.g., L232Cfs*9)⁹Amino acid position 233 (e.g., L233S)⁸⁶ Amino acid position 238 (e.g.,G238V)^(2,7) Amino acid position 242 (e.g., T242I)⁷ Amino acid position245 (e.g., I245Tfs*26)⁵⁷ Amino acid position 256 (e.g., A256G)⁹ Aminoacid position 260 (e.g., G260D)⁵⁷ Amino acid position 284 (e.g., V284L)⁷Amino acid position 297 (e.g., E297G)^(2,7) Amino acid position 303(e.g., R303K⁸, R303M⁶³, R303fsX321⁸³) Amino acid position 304 (e.g.,Y304X)²⁶ Amino acid position 312 (e.g., Q312H)⁷ Amino acid position 313(e.g., R313S)⁷ Amino acid position 314 (e.g., W314X)⁵⁷ Amino acidposition 318 (e.g., K318Rfs*26)²⁹ Amino acid position 327 (e.g., G327E)⁷Amino acid position 330 (e.g., V330X)²⁴ Amino acid position 336 (e.g.,C336S)^(2,7) Amino acid position 337 (e.g., Y337H)²¹ Amino acid position342 (e.g., W342G)⁵⁰ Amino acid position 354 (e.g., R354X)⁹ Amino acidposition 361 (e.g., Q361X)⁵⁷ Amino acid position 366 (e.g., V366D)⁵⁷Amino acid position 386 (e.g., G386X)³⁴ Δ Amino acid positions 383-389⁵⁷Amino acid position 387 (e.g., R387H)⁹ Amino acid position 390 (e.g.,A390P)⁷ Amino acid position 410 (e.g., G410D)⁷ Amino acid position 413(e.g., L413W)⁷ Amino acid position 415 (e.g., R415X)⁴² Amino acidposition 420 (e.g., I420T)⁹ Amino acid position 454 (e.g., V454X)⁴⁹Amino acid position 455 (e.g., G455E)⁹ Amino acid position 461 (e.g.,K461E)^(2,7) Amino acid position 463 (e.g., T463I)⁷ Amino acid position466 (e.g., Q466K)⁷ Amino acid position 470 (e.g., R470Q⁷, R470X⁹) Aminoacid position 472 (e.g., Y472X¹⁴, Y472C²⁷) Amino acid position 475(e.g., C475X)²⁹ Amino acid position 481 (e.g., V481E)⁷ Amino acidposition 482 (e.g., D482G)^(2,7) Amino acid position 484 (e.g.,H484Rfs*5)⁹ Amino acid position 487 (e.g., R487H², R487P⁸⁴) Amino acidposition 490 (e.g., N490D)⁷ Amino acid position 493 (e.g., W493X)⁸ Aminoacid position 498 (e.g., I498T)⁷ Amino acid position 501 (e.g., V501G)⁶⁸Amino acid position 512 (e.g., I512T)⁷ Amino acid position 515 (e.g.,N515T⁷, N515D⁶⁴) Amino acid position 516 (e.g., I516M)¹⁷ Amino acidposition 517 (e.g., R517H)⁷ Amino acid position 520 (e.g., R520X)⁵⁷Amino acid position 523 (e.g., A523G)¹³ Amino acid position 528 (e.g.,I528X)⁹ Amino acid position 540 (e.g., F540L)⁴⁶ Amino acid position 541(e.g., I541L⁷, I541T¹⁷) Amino acid position 548 (e.g., F548Y)⁷ Aminoacid position 549 (e.g., D549V)⁹ Amino acid position 554 (e.g., E554K)²¹Amino acid position 559 (e.g., M559T)⁵⁷ Amino acid position 562 (e.g.,G562D)⁷ Amino acid position 570 (e.g., A570T⁷, A570V²⁶) Amino acidposition 575 (e.g., R575X², R575Q²¹) Amino acid position 588 (e.g.,A588V)⁷ Amino acid position 591 (e.g., N591S)^(9,17) Amino acid position593 (e.g., S593R)^(2,7) Amino acid position 597 (e.g., V597V⁹, V597L¹³)Amino acid positions 591 and 597 (e.g., N591S + V597V)⁹ Amino acidposition 603 (e.g., K603K)⁵⁵ Amino acid position 609 (e.g.,H609Hfs*46)²⁶ Amino acid position 610 (e.g., I610Gfs*45)⁹ Amino acidposition 615 (e.g., H615R)²⁶ Amino acid position 625 (e.g., T625Nfs*5)²⁶Amino acid position 627 (e.g., I627T)⁷ Amino acid position 636 (e.g.,E636G)² Amino acid position 669 (e.g., I669V)²⁶ Amino acid position 698(e.g., R609H)⁹ Amino acid positions 112 and 698 (e.g., I112T + R698H)⁹Amino acid position 699 (e.g., S699P)⁹ Amino acid position 766 (e.g.,G766R)²⁴ Amino acid position 806 (e.g., G806G)⁵⁵ Amino acid position 824(e.g., G824E)⁴² Amino acid position 832 (e.g., R832C^(7,26), R832H⁴¹)Amino acid position 842 (e.g., D842G)² Amino acid position 859 (e.g.,T859R)⁷ Amino acid position 865 (e.g., A865V)⁴⁵ Amino acid position 877(e.g., G877R)⁵⁶ Amino acid position 893 (e.g., A893V)⁵⁷ Amino acidposition 901 (e.g., S901R)¹⁷ Amino acid position 903 (e.g., V903G)⁵⁷ ΔAmino acid position 919¹² Amino acid position 928 (e.g., R928X)^(15,21)Amino acid position 930 (e.g., K930Efs*79¹⁰, K930Efs*49²⁶) Amino acidposition 948 (e.g., R948C)^(7,26) Amino acid position 979 (e.g., N979D)⁷Amino acid position 982 (e.g., G982R)^(2,7) Amino acid positions 444 and982 (e.g., V444A + G982R)³⁸ Amino acid position 1001 (e.g., R1001R)⁹Amino acid position 1003 (e.g., G1003R)²⁴ Amino acid position 1004(e.g., G1004D)^(2,7) Amino acid position 1027 (e.g., S1027R)²⁶ Aminoacid position 1028 (e.g., A1028A)¹⁰ Amino acid position 1032 (e.g.,G1032R)¹² Amino acid position 1041 (e.g., Y1041X)⁹ Amino acid position1050 (e.g., R1050C)⁵⁷ Amino acid position 1053 (e.g., Q1053X)⁵⁷ Aminoacid position 1055 (e.g., L1055P)³⁶ Amino acid position 1057 (e.g.,R1057X)² Amino acid position 1058 (e.g., Q1058Hfs*38⁹, Q1058fs*38¹⁷)Amino acid position 1061 (e.g., I1061Vfs*34)⁹ Amino acid position 1083(e.g., C1083Y)⁴⁷ Amino acid position 1090 (e.g., R1090X)² Amino acidposition 1099 (e.g., L1099Lfs*38)²⁶ Amino acid position 1100 (e.g.,S1100Qfs*38)¹³ Amino acid position 1110 (e.g., A1110E)⁷ Amino acidposition 1116 (e.g., G1116R⁷, G1116F^(9,17), G1116E³⁶) Amino acidposition 1128 (e.g., R1128C)^(7,13) Amino acid position 1131 (e.g.,D1131V)²⁷ Amino acid position 1144 (e.g., S1144R)⁷ Amino acid position1153 (e.g., R1153C^(2,7), R1153H^(7,26)) Amino acid position 1154 (e.g.,S1154P)⁷ Amino acid position 1173 (e.g., N1173D)⁵⁷ Amino acid position1192 (e.g., A1192Efs*50)⁹ Amino acid position 1198 (e.g., H1198R)²⁷Amino acid position 1210 (e.g., T1210P⁷, T1210F⁵⁷) Amino acid position1211 (e.g., N1211D)⁷ Amino acid position 1212 (e.g., V1212F)³⁶ Aminoacid position 1231 (e.g., R1231W⁷, R1223Q⁷) Amino acid position 1232(e.g., A1232D)¹⁷ Amino acid position 1235 (e.g., R1235X)¹² Amino acidposition 1242 (e.g., L1242I)⁷ Amino acid position 1256 (e.g.,T1256fs*1296)⁸³ Amino acid position 1268 (e.g., R1268Q)^(2,7) Amino acidposition 1302 (e.g. E1302X)⁵⁷ Amino acid position 1311 (e.g., Y1311X)⁵⁷Amino acid position 1316 (e.g., T1316Lfs*64)¹⁵ Intron 4 ((+3)A > C)¹Splice site mutation IVS7 + 1G > A¹⁴ IVS8 + 1G > C⁷⁶ Splice sitemutation IVS9 + 1G > T¹⁴ Splice site mutation IVS13del − 13{circumflexover ( )} − 8¹⁴ Splice site mutation IVS16 − 8T > G¹⁴ Splice sitemutation IVS18 + 1G > A¹⁴ Splice site mutation IVS19 + 2T > C¹⁴ IVS 23 −8 G − A³⁶ IVS24 + 5G > A⁵¹ Putative splice mutation 1198 − 1G > C¹⁷Putative splice mutation 1810 − 3C > G¹⁷ Putative splice mutation 2178 +1G > A¹⁷ Putative splice mutation 2344 − 1G > T¹⁷ Putative splicemutation 3213 + 1_3213 + 2delinsA¹⁷ c.−24C > A⁷⁸ c.76 13 G > T⁹ c.77 −19T > A⁵² c.90_93delGAAA¹⁸ c.124G > A⁶⁹ c.150 + 3 A > C¹⁰c.249_250insT¹⁸ c.611 + 1G > A⁸⁴ c.611 + 4A > G³⁶ c.612 − 15_− 6del10bp⁵⁵ c.625A > C³¹ c.627 + 5G > T³¹ c.625A > C/c.627 + 5G > T³¹ c.886C >T³¹ c.890A > G⁵⁹ c.908 + 1G > A⁵⁷ c.908 + 5G > A⁵⁵ c.908delG⁵⁹ 1273 1 bpdeletion⁹¹ c.1084 − 2A > G⁵⁷ c.1445A > G⁵⁹ c.1587 − 1589delCTT³¹c.1621A > C⁵⁹ 1939delA¹⁴ c.2081T > A³¹ 2098delA¹⁶ c.2343 + 1 G > T⁸⁰c.2178 + 1G > T³⁶ c.2417G > A⁷⁸ c.2620C > T³² c.2815 − 8A > G⁵⁵c.3003A > G³⁷ c.3213 + 4 A > G^(9,37) c.3213 + 5 G > A⁹ c.3268C > T⁷⁵c.3382C > T⁷⁵ c.3765(+1 + 5)del5⁴² c.3767 − 3768insC⁶ 1145delC⁸Ex13_Ex17del⁸² ^(A) A mutation to ‘X’ denotes an early stop codon

REFERENCES FOR TABLES 4 AND 5

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In some embodiments, the mutation in ABCB11 is selected from A167T,G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V,G982R, R1153C, and R1268Q.

Provided are methods of treating PFIC (e.g., PFIC-1 and PFIC-2) in asubject that includes performing an assay on a sample obtained from thesubject to determine whether the subject has a mutation associated withPFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation), andadministering (e.g., specifically or selectively administering) atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof, to the subject determined tohave a mutation associated with PFIC. In some embodiments, the mutationis a ATP8B1 or ABCB11 mutation. For example, a mutation as provided inany one of Tables 1-4. In some embodiments, the mutation in ATP8B1 isselected from L127P, G308V, T456M, D554N, F529del, 661T, E665X, R930X,R952X, R1014X, and G1040R. In some embodiments, the mutation in ABCB11is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H,A570T, N591S, A865V, G982R, R1153C, and R1268Q.

Also provided are methods for treating PFIC (e.g., PFIC-1 and PFIC-2) ina subject in need thereof, the method comprising: (a) detecting amutation associated with PFIC (e.g., a ATP81, ABCB11, ABCB4, TJP2, NR1H4or Myo5b mutation) in the subject; and (b) administering to the subjecta therapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, methodsfor treating PFIC can include administering a therapeutically effectiveamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof, to a subject having a mutation associated with PFIC (e.g.,a ATP81, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation). In someembodiments, the mutation is a ATP8B1 or ABCB11 mutation. For example, amutation as provided in any one of Tables 1-4. In some embodiments, themutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del,1661T, E665X, R930X, R952X, R1014X, and G1040R. In some embodiments, themutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q,R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.

In some embodiments, the subject is determined to have a mutationassociated with PFIC in a subject or a biopsy sample from the subjectthrough the use of any art recognized tests, including next generationsequencsing (NGS). In some embodiments, the subject is determined tohave a mutation associated with PFIC using a regulatory agency-approved,e.g., FDA-approved test or assay for identifying a mutation associatedwith PFIC in a subject or a biopsy sample from the subject or byperforming any of the non-limiting examples of assays described herein.Additional methods of diagnosing PFIC are described in Gunaydin, M. etal., Hepat Med. 2018, vol. 10, p. 95-104, incorporated by reference inits entirety herein.

In some embodiments, the treatment of PFIC (e.g., PFIC-1 or PFIC-2)decreases the level of serum bile acids in the subject. In someembodiments, the level of serum bile acids is determined by, forexample, an ELISA enzymatic assay or the assays for the measurement oftotal bile acids as described in Danese et al., PLoS One. 2017, vol.12(6): e0179200, which is incorporated by reference herein in itsentirety. In some embodiments, the level of serum bile acids candecrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to70%, 50% to 80%, or by more than 90% of the level of serum bile acidsprior to administration of a compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, thetreatment of PFIC includes treatment of pruritus.

Since LBAT is expressed on hepatocytes, LBAT and dual ASBT/LBATinhibitor substances need to have at least some bioavailability and freefraction in blood. Because LBAT inhibitor compounds only need to survivefrom the intestine to the liver, it is expected that a relatively lowsystemic exposure of such compounds will be sufficient, therebyminimizing the potential risk for any side effects in the rest of thebody. It is expected that inhibition of LBAT and ASBT will have at leastadditive effects in decreasing the intrahepatic bile acid concentration.It is also expected that a dual ASBT/LBAT inhibitor may be able toreduce bile acid levels without inducing diarrhoea, as is sometimesobserved with ASBT inhibitors.

Compounds having a high LBAT inhibiting potency and sufficientbioavailability are expected to be particularly suitable for thetreatment of hepatitis. Compounds having a dual ASBT/LBAT inhibitingpotency and sufficient bioavailability are expected to be particularlysuitable for the treatment of non-alcoholic steatohepatitis (NASH).

NASH is a common and serious chronic liver disease that resemblesalcoholic liver disease, but that occurs in people who drink little orno alcohol. In NASH patients, fat accumulation in the liver, known asnonalcoholic fatty liver disease (NAFLD) or steatosis, and other factorssuch as high LDL cholesterol and insulin resistance induce chronicinflammation in the liver and may lead to progressive scarring oftissue, known as fibrosis, and cirrhosis, followed eventually by liverfailure and death. Patients with NASH have been found to havesignificantly higher total serum bile acid concentrations than healthysubjects under fasting conditions (2.2- to 2.4-fold increase in NASH)and at all post-prandial time points (1.7- to 2.2-fold increase inNASH). These are driven by increased taurine- and glycine-conjugatedprimary and secondary bile acids. Patients with NASH exhibited greatervariability in their fasting and post-prandial bile acid profile. Theseresults indicate that patients with NASH have higher fasting andpost-prandial exposure to bile acids, including the more hydrophobic andcytotoxic secondary species. Increased bile acid exposure may beinvolved in liver injury and the pathogenesis of NAFLD and NASH (Ferslewet al., Dig Dis Sci. 2015, vol. 60, p. 3318-3328). It is thereforelikely that ASBT and/or LBAT inhibition will be beneficial for thetreatment of NASH.

NAFLD is characterized by hepatic steatosis with no secondary causes ofhepatic steatosis including excessive alcohol consumption, other knownliver diseases, or long-term use of a steatogenic medication (Chalasaniet al., Hepatology 2018, vol. 67(1), p. 328-357). NAFLD can becategorized into non-alcoholic fatty liver (NAFL) and non-alcoholicsteatohepatitis (NASH). According to Chalasani et al., NAFL is definedas the presence of 5% hepatic steatosis without evidence ofhepatocellular injury in the form of hepatocyte ballooning. NASH isdefined as the presence of 5% hepatic steatosis and inflammation withhepatocyte injury (e.g., ballooning), with or without any liverfibrosis. NASH is also commonly associated with hepatic inflammation andliver fibrosis, which can progress to cirrhosis, end-stage liverdisease, and hepatocellular carcinoma. While liver fibrosis is notalways present in NASH, the severity of the fibrosis, when present, canbe linked to long-term outcomes.

There are many approaches used to assess and evaluate whether a subjecthas NAFLD and if so, the severity of the disease, includingdifferentiating whether the NAFLD is NAFL or NASH. In some embodiments,the severity of NAFLD can be assessed using the NAS. In someembodiments, treatment of NAFLD can be assessed using the NAS. In someembodiments, the NAS can be determined as described in Kleiner et al.,Hepatology. 2005, 41(6):1313-1321, which is hereby incorporated byreference in its entirety. See, for example, Table 6 for a simplifiedNAS scheme adapted from Kleiner.

TABLE 6 Example of the NAFLD Activity Score (NAS) with Fibrosis StageFeature Degree Score Steatosis     <5% 0  5-33% 1 >33-66% 2    >66% 3Lobular No foci 0 Inflammation <2 foci/200x 1 2-4 foci/200x  2 >4foci/200x 3 Ballooning None 0 degeneration Few 1 Many cells/Prominent 2ballooning Fibrosis None 0 Perisinusoidal or 1 periportal Perisinusoidal& 2 portal/periportal Bridging fibrosis 3 Cirrhosis 4

In some embodiments, the NAS is determined non-invasively, for example,as described in U.S. Application Publication No. 2018/0140219, which isincorporated by reference herein in its entirety. In some embodiments,the NAS is determined for a sample from the subject prior toadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the NAS is determinedduring the period of time or after the period of time of administrationof a compound of formula (I), or a pharmaceutically acceptable saltthereof. In some embodiments, a lower NAS score during the period oftime or after the period of time of administration of a compound offormula (I), or a pharmaceutically acceptable salt thereof compared toprior to administration of the compound of formula (I), or apharmaceutically acceptable salt thereof indicates treatment of NAFLD(e.g., NASH). For example, a decrease in the NAS by 1, by 2, by 3, by 4,by 5, by 6, or by 7 indicates treatment of NAFLD (e.g., NASH). In someembodiments, the NAS following administration of a compound of formula(I), or a pharmaceutically acceptable salt thereof, is 7 or less. Insome embodiments, the NAS during the period of time of administration ofa compound of formula (I), or a pharmaceutically acceptable saltthereof, is 5 or less, 4 or less, 3 or less, or 2 or less. In someembodiments, the NAS during the period of time of administration of acompound of formula (I), or a pharmaceutically acceptable salt thereof,is 7 or less. In some embodiments, the NAS during the period of time ofadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is 5 or less, 4 or less, 3 or less, or 2 orless. In some embodiments, the NAS after the period of time ofadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is 7 or less. In some embodiments, the NASafter the period of time of administration of a compound of formula (I),or a pharmaceutically acceptable salt thereof, is 5 or less, 4 or less,3 or less, or 2 or less.

Additional approaches of assessing and evaluating NASH in a subjectinclude determining one or more of hepatic steatosis (e.g., accumulationof fat in the liver); hepatic inflammation; biomarkers indicative of oneor more of liver damage, hepatic inflammation, liver fibrosis, and/orliver cirrhosis (e.g., serum markers and panels). Further examples ofphysiological indicators of NASH can include liver morphology, liverstiffness, and the size or weight of the subject's liver.

In some embodiments, NASH in the subject is evidenced by an accumulationof hepatic fat and detection of a biomarker indicative of liver damage.For example, elevated serum ferritin and low titers of serumautoantibodies can be common features of NASH.

In some embodiments, methods to assess NASH include magnetic resonanceimaging, either by spectroscopy or by proton density fat fraction(MRI-PDFF) to quantify steatosis, transient elastography (FIBROSCAN®),hepatic venous pressure gradient (HPVG), hepatic stiffness measurementwith MRE for diagnosing significant liver fibrosis and/or cirrhosis, andassessing histological features of liver biopsy. In some embodiments,magnetic resonance imaging is used to detect one or more ofsteatohepatitis (NASH-MRI), liver fibrosis (Fibro-MRI), and steatosis.See, for example, U.S. Application Publication Nos. 2016/146715 and2005/0215882, each of which are incorporated herein by reference intheir entireties.

In some embodiments, treatment of NASH can include a decrease of one ormore symptoms associated with NASH; reduction in the amount of hepaticsteatosis; a decrease in the NAS; a decrease in hepatic inflammation; adecrease in the level of biomarkers indicative of one or more of liverdamage, inflammation, liver fibrosis, and/or liver cirrhosis; and areduction in fibrosis and/or cirrhosis, a lack of further progression offibrosis and/or cirrhosis, or a slowing of the progression of fibrosisand/or cirrhosis in the subject following administration of one or moredoses of a compound of formula (I), or a pharmaceutically acceptablesalt thereof.

In some embodiments, treatment of NASH comprises a decrease of one ormore symptoms associated with NASH in the subject. Exemplary symptomscan include one or more of an enlarged liver, fatigue, pain in the upperright abdomen, abdominal swelling, enlarged blood vessels just beneaththe skin's surface, enlarged breasts in men, enlarged spleen, red palms,jaundice, and pruritus. In some embodiments, the subject isasymptomatic. In some embodiments, the total body weight of the subjectdoes not increase. In some embodiments, the total body weight of thesubject decreases. In some embodiments, the body mass index (BMI) of thesubject does not increase. In some embodiments, the body mass index(BMI) of the subject decreases. In some embodiments, the waist and hip(WTH) ratio of the subject does not increase. In some embodiments, thewaist and hip (WTH) ratio of the subject decreases.

In some embodiments, treatment of NASH can be assessed by measuringhepatic steatosis. In some embodiments, treatment of NASH comprises areduction in hepatic steatosis following administration of a compound offormula (I), or a pharmaceutically acceptable salt thereof, as describedherein. In some embodiments, hepatic steatosis is determined by one ormore methods selected from the group consisting of ultrasonography,computed tomography (CT), magnetic resonance imaging, magnetic resonancespectroscopy (MRS), magnetic resonance elastography (MRE), transientelastography (TE) (e.g., FIBROSCAN®), measurement of liver size orweight, or by liver biopsy (see, e.g., Di Lascio et al., Ultrasound MedBiol. 2018, vol. 44(8), p. 1585-1596; Lv et al., J Clin Transl Hepatol.2018, vol. 6(2), p. 217-221; Reeder et al., J Magn Reson Imaging. 2011,vol. 34(4), spcone; and de Ledinghen V, et al., J Gastroenterol Hepatol.2016, vol. 31(4), p. 848-855, each of which are incorporated herein byreference in their entireties). A subject diagnosed with NASH can havegreater than about 5% hepatic steatosis, for example, greater than about5% to about 25%, about 25% to about 45%, about 45% to about 65%, orgreater than about 65% hepatic steatosis. In some embodiments, a subjectwith greater than about 5% to about 33% hepatic steatosis has stage 1hepatic steatosis, a subject with about 33% to about 66% hepaticsteatosis has stage 2 hepatic steatosis, and a subject with greater thanabout 66% hepatic steatosis has stage 3 hepatic steatosis.

In some embodiments, the amount of hepatic steatosis is determined priorto administration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the amount of hepaticsteatosis is determined during the period of time or after the period oftime of administration of the compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, areduction in the amount of hepatic steatosis during the period of timeor after the period of time of administration of the compound of formula(I), or a pharmaceutically acceptable salt thereof, compared to prior toadministration of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, indicates treatment of NASH. For example, areduction in the amount of hepatic steatosis by about 1% to about 50%,about 25% to about 75%, or about 50% to about 100% indicates treatmentof NASH. In some embodiments, a reduction in the amount of hepaticsteatosis by about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% indicates treatment of NASH.

In some embodiments, the presence of hepatic inflammation is determinedby one or more methods selected from the group consisting of biomarkersindicative of hepatic inflammation and a liver biopsy sample(s) from thesubject. In some embodiments, the severity of hepatic inflammation isdetermined from a liver biopsy sample(s) from the subject. For example,hepatic inflammation in a liver biopsy sample can be assessed asdescribed in Kleiner et al., Hepatology 2005, vol. 41(6), p. 1313-1321and Brunt et al., Am J Gastroenterol 1999, vol. 94, p. 2467-2474, eachof which are hereby incorporated by reference in their entireties. Insome embodiments, the severity of hepatic inflammation is determinedprior to administration of a compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, theseverity of hepatic inflammation is determined during the period of timeor after the period of time of administration of a compound of formula(I), or a pharmaceutically acceptable salt thereof. In some embodiments,a decrease in the severity of hepatic inflammation during the period oftime or after the period of time of administration of a compound offormula (I), or a pharmaceutically acceptable salt thereof, compared toprior to administration of the compound of formula (I), or apharmaceutically acceptable salt thereof, indicates treatment of NASH.For example, a decrease in the severity of hepatic inflammation by about1% to about 50%, about 25% to about 75%, or about 50% to about 100%indicates treatment of NASH. In some embodiments, a decrease in theseverity of hepatic inflammation by about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% indicates treatment of NASH.

In some embodiments, treatment of NASH comprises treatment of fibrosisand/or cirrhosis, e.g., a decrease in the severity of fibrosis, a lackof further progression of fibrosis and/or cirrhosis, or a slowing of theprogression of fibrosis and/or cirrhosis. In some embodiments, thepresence of fibrosis and/or cirrhosis is determined by one or moremethods selected from the group consisting of transient elastography(e.g., FIBROSCAN®), non-invasive markers of hepatic fibrosis, andhistological features of a liver biopsy. In some embodiments, theseverity (e.g., stage) of fibrosis is determined by one or more methodsselected from the group consisting of transient elastography (e.g.,FIBROSCAN®), a fibrosis-scoring system, biomarkers of hepatic fibrosis(e.g., non-invasive biomarkers), and hepatic venous pressure gradient(HVPG). Non-limiting examples of fibrosis scoring systems include theNAFLD fibrosis scoring system (see, e.g., Angulo et al., Hepatology2007, vol. 45(4), p. 846-54), the fibrosis scoring system in Brunt etal., Am. J. Gastroenterol. 1999, vol. 94, p. 2467-2474, the fibrosisscoring system in Kleiner et al., Hepatology 2005, vol. 41(6), p.1313-1321, and the ISHAK fibrosis scoring system (see Ishak et al., J.Hepatol. 1995, vol. 22, p. 696-699), the contents of each of which areincorporated by reference herein in their entireties.

In some embodiments, the severity of fibrosis is determined prior toadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the severity of fibrosisis determined during the period of time or after the period of time ofadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, a decrease in the severityof fibrosis during the period of time or after the period of time ofadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, compared to prior to administration of thecompound of formula (I), or a pharmaceutically acceptable salt thereof,indicates treatment of NASH. In some embodiments, a decrease in theseverity of fibrosis, a lack of further progression of fibrosis and/orcirrhosis, or a slowing of the progression of fibrosis and/or cirrhosisindicates treatment of NASH. In some embodiments, the severity offibrosis is determined using a scoring system such as any of thefibrosis scoring systems described herein, for example, the score canindicate the stage of fibrosis, e.g., stage 0 (no fibrosis), stage 1,stage 2, stage 3, and stage 4 (cirrhosis) (see, e.g., Kleiner et al). Insome embodiments, a decrease in the stage of the fibrosis is a decreasein the severity of the fibrosis. For example, a decrease by 1, 2, 3, or4 stages is a decrease in the severity of the fibrosis. In someembodiments, a decrease in the stage, e.g., from stage 4 to stage 3,from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 tostage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage1 to stage 0 indicates treatment of NASH. In some embodiments, the stageof fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2,from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 tostage 1, from stage 2 to stage 0, or from stage 1 to stage 0 followingadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, compared to prior to administration of thecompound of formula (I), or a pharmaceutically acceptable salt thereof.In some embodiments, the stage of fibrosis decreases from stage 4 tostage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or fromstage 1 to stage 0 during the period of time of administration of acompound of formula (I), or a pharmaceutically acceptable salt thereof,compared to prior to administration of the compound of formula (I), or apharmaceutically acceptable salt thereof. In some embodiments, the stageof fibrosis decreases from stage 4 to stage 3, from stage 4 to stage 2,from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 tostage 1, from stage 2 to stage 0, or from stage 1 to stage 0 after theperiod of time of administration of a compound of formula (I), or apharmaceutically acceptable salt thereof, compared to prior toadministration of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the presence of NASH is determined by one or morebiomarkers indicative of one or more of liver damage, inflammation,liver fibrosis, and/or liver cirrhosis or scoring systems thereof. Insome embodiments, the severity of NASH is determined by one or morebiomarkers indicative of one or more of liver damage, inflammation,liver fibrosis, and/or liver cirrhosis or scoring systems thereof. Thelevel of the biomarker can be determined by, for example, measuring,quantifying, and monitoring the expression level of the gene or mRNAencoding the biomarker and/or the peptide or protein of the biomarker.Non-limiting examples of biomarkers indicative of one or more of liverdamage, inflammation, liver fibrosis, and/or liver cirrhosis and/orscoring systems thereof include the aspartate aminotransferase (AST) toplatelet ratio index (APRI); the aspartate aminotransferase (AST) andalanine aminotransferase (ALT) ratio (AAR); the FIB-4 score, which isbased on the APRI, alanine aminotransferase (ALT) levels, and age of thesubject (see, e.g., McPherson et al., Gut 2010, vol. 59(9), p. 1265-9,which is incorporated by reference herein in its entirety); hyaluronicacid; pro-inflammatory cytokines; a panel of biomarkers consisting ofα2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gammaglutamyl transpeptidase (GGT) combined with a subject's age and genderto generate a measure of fibrosis and necroinflammatory activity in theliver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consistingof bilirubin, gamma-glutamyltransferase, hyaluronic acid,α2-macroglobulin combined with the subject's age and sex (e.g.,HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol. 51(10), p.1867-1873), and a panel of biomarkers consisting of tissue inhibitor ofmetalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g.,FIBROSPECT®); a panel of biomarkers consisting of tissue inhibitor ofmetalloproteinases 1 (TIMP-1), amino-terminal propeptide of type IIIprocollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced LiverFibrosis (ELF) score, see, e.g., Lichtinghagen R, et al., J Hepatol.2013 August; 59(2):236-42, which is incorporated by reference herein inits entirety). In some embodiments, the presence of fibrosis isdetermined by one or more of the FIB-4 score, a panel of biomarkersconsisting of α2-macroglobulin, haptoglobin, apolipoprotein A1,bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject'sage and gender to generate a measure of fibrosis and necroinflammatoryactivity in the liver (e.g., FIBROTEST®, FIBROSURE®), a panel ofbiomarkers consisting of bilirubin, gamma-glutamyltransferase,hyaluronic acid, α2-macroglobulin combined with the subject's age andsex (e.g., HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol.51(10), p. 1867-1873), and a panel of biomarkers consisting of tissueinhibitor of metalloproteinase-1, hyaluronic acid, and α2-macroglobulin(e.g., FIBROSPECT®); and a panel of biomarkers consisting of tissueinhibitor of metalloproteinases 1 (TIMP-1), amino-terminal propeptide oftype III procollagen (PIIINP) and hyaluronic acid (HA) (e.g., theEnhanced Liver Fibrosis (ELF) score). In some embodiments, the level ofaspartate aminotransferase (AST) does not increase. In some embodiments,the level of aspartate aminotransferase (AST) decreases. In someembodiments, the level of alanine aminotransferase (ALT) does notincrease. In some embodiments, the level of alanine aminotransferase(ALT) decreases. In some embodiments, the “level” of an enzyme refers tothe concentration of the enzyme, e.g., within blood. For example, thelevel of AST or ALT can be expressed as Units/L.

In some embodiments, the severity of fibrosis is determined by one ormore of the FIB-4 score, a panel of biomarkers consisting ofα2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gammaglutamyl transpeptidase (GGT) combined with a subject's age and genderto generate a measure of fibrosis and necroinflammatory activity in theliver (e.g., FIBROTEST®, FIBROSURE®), a panel of biomarkers consistingof bilirubin, gamma-glutamyltransferase, hyaluronic acid,α2-macroglobulin combined with the subject's age and sex (e.g.,HEPASCORE®; see, e.g., Adams et al., Clin. Chem. 2005, vol. 51(10), p.1867-1873, which is incorporated by reference herein in its entirety),and a panel of biomarkers consisting of tissue inhibitor ofmetalloproteinase-1, hyaluronic acid, and α2-macroglobulin (e.g.,FIBROSPECT®); and a panel of biomarkers consisting of tissue inhibitorof metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type IIIprocollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced LiverFibrosis (ELF) score).

In some embodiments, hepatic inflammation is determined by the level ofliver inflammation biomarkers, e.g., pro-inflammatory cytokines.Non-limiting examples of biomarkers indicative of liver inflammationinclude interleukin-(IL) 6, interleukin-(IL) 1β, tumor necrosis factor(TNF)-α, transforming growth factor (TGF)-β, monocyte chemotacticprotein (MCP)-1, C-reactive protein (CRP), PAI-1, and collagen isoformssuch as Col1a1, Col1a2, and Col4a1 (see, e.g., Neuman, et al., Can. J.Gastroenterol. Hepatol. 2014, vol. 28(11), p. 607-618 and U.S. Pat. No.9,872,844, each of which are incorporated by reference herein in theirentireties). Liver inflammation can also be assessed by change ofmacrophage infiltration, e.g., measuring a change of CD68 expressionlevel. In some embodiments, liver inflammation can be determined bymeasuring or monitoring serum levels or circulating levels of one ormore of interleukin-(IL) 6, interleukin-(IL) 1P, tumor necrosis factor(TNF)-α, transforming growth factor (TGF)-β, monocyte chemotacticprotein (MCP)-1, and C-reactive protein (CRP).

In some embodiments, the level of one or more biomarkers indicative ofone or more of liver damage, inflammation, liver fibrosis, and/or livercirrhosis is determined for a sample from the subject prior toadministration of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the level of one or morebiomarkers indicative of one or more of liver damage, inflammation,liver fibrosis, and/or liver cirrhosis is determined during the periodof time or after the period of time of administration of a compound offormula (I), or a pharmaceutically acceptable salt thereof. In someembodiments, a decrease in the level of one or more biomarkersindicative of one or more of liver damage, inflammation, liver fibrosis,and/or liver cirrhosis during the period of time or after the period oftime of administration of a compound of formula (I), or apharmaceutically acceptable salt thereof, compared to prior toadministration of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, indicates treatment of NASH. For example, adecrease in the level of one or more biomarkers indicative of one ormore of liver damage, inflammation, liver fibrosis, and/or livercirrhosis by at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 99% indicatestreatment of NASH. In some embodiments, the decrease in the level of oneor more biomarkers indicative of one or more of liver damage,inflammation, liver fibrosis, and/or liver cirrhosis followingadministration of the compound of formula (I), or a pharmaceuticallyacceptable salt thereof, is by at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 99%. In some embodiments, the level of one or more biomarkersindicative of one or more of liver damage, inflammation, liver fibrosis,and/or liver cirrhosis during the period of time of administration of acompound of formula (I), or a pharmaceutically acceptable salt thereof,is by at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 99%. In someembodiments, the level of one or more biomarkers indicative of one ormore of liver damage, inflammation, liver fibrosis, and/or livercirrhosis after the period of time of administration of a compound offormula (I), or a pharmaceutically acceptable salt thereof, is by atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99%.

In some embodiments, the treatment of NASH decreases the level of serumbile acids in the subject. In some embodiments, the level of serum bileacids is determined by, for example, an ELISA enzymatic assay or theassays for the measurement of total bile acids as described in Danese etal., PLoS One. 2017, vol. 12(6): e0179200, which is incorporated byreference herein in its entirety. In some embodiments, the level ofserum bile acids can decrease by, for example, 10% to 40%, 20% to 50%,30% to 60%, 40% to 70%, 50% to 80%, or by more than 90% of the level ofserum bile acids prior to administration of a compound of formula (I),or a pharmaceutically acceptable salt thereof. In some embodiments, theNASH is NASH with attendant cholestasis. In cholestasis, the release ofbile, including bile acids, from the liver is blocked. Bile acids cancause hepatocyte damage (see, e.g., Perez M J, Briz O. World J.Gastroenterol. 2009, vol. 15(14), p. 1677-1689) likely leading to orincreasing the progression of fibrosis (e.g., cirrhosis) and increasingthe risk of hepatocellular carcinoma (see, e.g., Sorrentino P et al.,Dig. Dis. Sci. 2005, vol. 50(6), p. 1130-1135 and Satapathy S K andSanyal A J. Semin. Liver Dis. 2015, vol. 35(3), p. 221-235, each ofwhich are incorporated by reference herein in their entireties). In someembodiments, the treatment of NASH includes treatment of pruritus. Insome embodiments, the treatment of NASH with attendant cholestasisincludes treatment of pruritus. In some embodiments, a subject with NASHwith attendant cholestasis has pruritus.

Exemplary biomarkers for NASH are provided in Table 7.

TABLE 7 Exemplary NASH biomarkers Liver Fibrosis Biomarkers Aspartateaminotransferase (AST) to platelet ratio index (APRI) Aspartateaminotransferase (AST) and alanine aminotransferase (ALT) ratio (AAR)FIB-4 score¹ Hyaluronic acid Pro-inflammatory cytokines A panelincluding α2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin,gamma glutamyl transpeptidase (GGT) combined with a subject's age andgender to generate a measure of fibrosis and necroinflammatory activityin the liver (e.g., FIBROTEST ®, FIBROSURE ®) A panel includingbilirubin, gamma-glutamyltransferase, hyaluronic acid, α2-macroglobulincombined with the subject's age and sex (e.g., HEPASCORE ®²) A panelincluding tissue inhibitor of metalloproteinase-1, hyaluronic acid, andα2-macroglobulin (e.g., FIBROSPECT ®) A panel including tissue inhibitorof metalloproteinases 1 (TIMP-1), amino-terminal propeptide of type IIIprocollagen (PIIINP) and hyaluronic acid (HA) (e.g., the Enhanced LiverFibrosis (ELF) score³) Liver inflammation biomarkers^(4,5)Interleukin-(IL) 6 Interleukin-(IL) 1β Tumor necrosis factor (TNF)-αTransforming growth factor (TGF)-β Monocyte chemotactic protein (MCP)-1C-reactive protein (CRP) PAI-1 Collagen isoforms (e.g., Col1a1, Col1a2,and Col4a1) Change of macrophage infiltration (e.g., a change of CD68expression level) References for Table 7 ¹McPherson et al., Gut. 2010,vol. 59(9), p. 1265-1269. ²Adams, et al. Clin Chem. 2005, vol. 51(10),p. 1867-1873. ³Lichtinghagen, et al. J Hepatol. 2013, vol. 59(2), p.236-242. ⁴Neuman, et al. Can J Gastroenterol Hepatol. 2014, vol. 28(11),p. 607-618. ⁵U.S. Pat. No. 9,872,844

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may show a higher free fraction in plasma. In some embodiments,the free fraction is greater than about 0.2%, such as greater than about0.4%, such as greater than about 0.6%, such as greater than about 0.8%,such as greater than about 1.0%, such as greater than about 1.25%, suchas greater than about 1.5%, such as greater than about 1.75%, such asgreater than about 2.0%, such as greater than about 2.5%, such asgreater than about 3%, such as greater than about 4%, such as greaterthan about 5%, such as greater than about 7.5%, such as greater thanabout 10%, or such as greater than about 20%.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may be excreted in urine. In some embodiments, the fraction ofthe compound that is excreted in urine is greater than about 0.2%, suchas greater than about 0.4%, such as greater than about 0.6%, such asgreater than about 0.8%, such as greater than about 1.0%, such asgreater than about 2%, such as greater than about 3%, such as greaterthan about 5%, such as greater than about 7.5%, such as greater thanabout 10%, such as greater than about 15%, such as greater than about20%, such as greater than about 30%, or such as greater than about 50%.

Following absorption from the intestine, some compounds of formula (I),or pharmaceutically acceptable salts thereof, may be circulated via theenterohepatic circulation. In some embodiments, the fraction of thecompound that is circulated via the enterohepatic circulation is greaterthan about 0.1%, such as greater than about 0.2%, such as greater thanabout 0.3%, such as greater than about 0.5%, such as greater than about1.0%, such as greater than about 1.5%, such as greater than about 2%,such as greater than about 3%, such as greater than about 5%, such asgreater than about 7%, such as greater than about 10%, such as greaterthan about 15%, such as greater than about 20%, such as greater thanabout 30% or such as greater than about 50%.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may cause renal excretion of bile salts. In some embodiments,the fraction of circulating bile acids that is excreted by the renalroute is greater than about 1%, such as greater than about 2%, such asgreater than about 5%, such as greater than about 7%, such as greaterthan about 10%, such as greater than about 15%, such as greater thanabout 20%, or such as greater than about 25%.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may show improved or optimal permeability. The permeability maybe measured in Caco2 cells, and values are given as Papp (apparentpermeability) values in cm/s. In some embodiments, the permeability isgreater than at least about 0.1×10⁻⁶ cm/s, such as greater than about0.2×10⁻⁶ cm/s, such as greater than about 0.4×10⁻⁶ cm/s, such as greaterthan about 0.7×10⁻⁶ cm/s, such as greater than about 1.0×10⁻⁶ cm/s, suchas greater than about 2×10⁻⁶ cm/s, such as greater than about 3×10⁻⁶cm/s, such as greater than about 5×10⁻⁶ cm/s, such as greater than about7×10⁻⁶ cm/s, such as greater than about 10×10⁻⁶ cm/s, such as greaterthan about 15×10⁻⁶ cm/s.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may show an improved or optimal bioavailability. In someembodiments, the oral bioavailability is greater than about 5%, such asgreater than about 7%, such as greater than about 10%, such as greaterthan about 15%, such as greater than about 20%, such as greater thanabout 30%, such as greater than about 40%, such as greater than about50%, such as greater than about 60%, such as greater than about 70% orsuch as greater than about 80%. In other embodiments, the oralbioavailability is between about 10 and about 90%, such as between about20 and about 80%, such as between about 30 and about 70% or such asbetween about 40 and about 60%.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may be a substrate to relevant transporters in the kidney.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may give rise to concentrations of bile acids in the intestine,the liver and in serum that do not cause adverse gastrointestinaleffects.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may decrease the concentration of bile acids in the liverwithout causing gastrointestinal disorders such as diarrhoea.

As used herein, the terms “treatment”, “treat” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

A suitable pharmaceutically acceptable salt of a compound of theinvention is, for example, a base-addition salt of a compound of theinvention which is sufficiently acidic, such as an alkali metal salt(e.g., a sodium or potassium salt), an alkaline earth metal salt (e.g.,a calcium or magnesium salt), an ammonium salt, or a salt with anorganic base which affords a physiologically acceptable cation, forexample a salt with methylamine, dimethylamine, trimethylamine,piperidine, morpholine or tris-(2-hydroxyethyl)amine.

Some compounds of formula (I), or pharmaceutically acceptable saltsthereof, may have chiral centres and/or geometric isomeric centres (E-and Z-isomers). It is to be understood that the invention encompassesall such optical isomers, diastereoisomers and geometric isomers thatpossess ASBT and/or LBAT inhibitory activity. The invention alsoencompasses any and all tautomeric forms of compounds of formula (I), orpharmaceutically acceptable salts thereof, that possess ASBT and/or LBATinhibitory activity. Certain compounds of formula (I), orpharmaceutically acceptable salts thereof, may exist in unsolvated aswell as solvated forms, such as, for example, hydrated forms. It is tobe understood that the invention encompasses all such solvated formsthat possess ASBT and/or LBAT inhibitory activity.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients. The excipients may e.g. includefillers, binders, disintegrants, glidants and lubricants. In general,pharmaceutical compositions may be prepared in a conventional mannerusing conventional excipients.

Examples of suitable fillers include, but are not limited to, dicalciumphosphate dihydrate, calcium sulfate, lactose (such as lactosemonohydrate), sucrose, mannitol, sorbitol, cellulose, microcrystallinecellulose, dry starch, hydrolyzed starches and pregelatinized starch. Incertain embodiments, the filler is mannitol and/or microcrystallinecellulose.

Examples of suitable binders include, but are not limited to, starch,pregelatinized starch, gelatin, sugars (such as sucrose, glucose,dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural andsynthetic gums (such as acacia gum and tragacanth gum), sodium alginate,cellulose derivatives (such as hydroxypropylmethylcellulose (orhypromellose), hydroxypropylcellulose and ethylcellulose) and syntheticpolymers (such as acrylic acid and methacrylic acid copolymers,methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkylmethacrylate copolymers, polyacrylic acid/polymethacrylic acidcopolymers and polyvinylpyrrolidone (povidone)). In certain embodiments,the binder is hydroxypropylmethylcellulose (hypromellose).

Examples of suitable disintegrants include, but are not limited to, drystarch, modified starch (such as (partially) pregelatinized starch,sodium starch glycolate and sodium carboxymethyl starch), alginic acid,cellulose derivatives (such as sodium carboxymethylcellulose,hydroxypropyl cellulose, and low substituted hydroxypropyl cellulose(L-HPC)) and cross-linked polymers (such as carmellose, croscarmellosesodium, carmellose calcium and cross-linked PVP (crospovidone)). Incertain embodiments, the disintegrant is croscarmellose sodium.

Examples of suitable glidants and lubricants include, but are notlimited to, talc, magnesium stearate, calcium stearate, stearic acid,glyceryl behenate, colloidal silica, aqueous silicon dioxide, syntheticmagnesium silicate, fine granulated silicon oxide, starch, sodium laurylsulfate, boric acid, magnesium oxide, waxes (such as carnauba wax),hydrogenated oil, polyethylene glycol, sodium benzoate, polyethyleneglycol, and mineral oil. In certain embodiments, the glidant orlubricant is magnesium stearate or colloidal silica.

The pharmaceutical composition may be conventionally coated with one ormore coating layers. Enteric coating layers or coating layers fordelayed or targeted release of the compound of formula (I), orpharmaceutically acceptable salts thereof, are also contemplated. Thecoating layers may comprise one or more coating agents, and mayoptionally comprise plasticizers and/or pigments (or colorants).

Example of suitable coating agents include, but are not limited to,cellulose-based polymers (such as ethylcellulose,hydroxypropylmethylcellulose (or hypromellose), hydroxypropylcellulose,cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose acetate succinate and hydroxypropyl methylcellulosephthalate), vinyl-based polymers (such as polyvinyl alcohol) andpolymers based on acrylic acid and derivatives thereof (such as acrylicacid and methacrylic acid copolymers, methacrylic acid copolymers,methyl methacrylate copolymers, aminoalkyl methacrylate copolymers,polyacrylic acid/polymethacrylic acid copolymers). In certainembodiments, the coating agent is hydroxypropylmethylcellulose. In otherembodiments, the coating agent is polyvinyl alcohol. Examples ofsuitable plasticizers include, but are not limited to, triethyl citrate,glyceryl triacetate, tributyl citrate, diethyl phthalate, acetyltributyl citrate, dibutyl phthalate, dibutyl sebacate and polyethyleneglycol. In certain embodiments, the plasticizer is polyethylene glycol.

Examples of suitable pigments include, but are not limited to, titaniumdioxide, iron oxides (such as yellow, brown, red or black iron oxides)and barium sulfate.

The pharmaceutical composition may be in a form that is suitable fororal administration, for parenteral injection (including intravenous,subcutaneous, intramuscular and intravascular injection), for topicaladministration of for rectal administration. In a preferred embodiment,the pharmaceutical composition is in a form that is suitable for oraladministration, such as a tablet or a capsule.

The dosage required for the therapeutic or prophylactic treatment willdepend on the route of administration, the severity of the disease, theage and weight of the patient and other factors normally considered bythe attending physician, when determining the appropriate regimen anddosage level for a particular patient.

The amount of the compound to be administered will vary for the patientbeing treated, and may vary from about 1 μg/kg of body weight to about50 mg/kg of body weight per day. A unit dose form, such as a tablet orcapsule, will usually contain about 1 to about 250 mg of activeingredient, such as about 1 to about 100 mg, or such as about 1 to about50 mg, or such as about 1 to about 20 mg, e.g. about 2.5 mg, or about 5mg, or about 10 mg, or about 15 mg. The daily dose can be administeredas a single dose or divided into one, two, three or more unit doses. Anorally administered daily dose of a bile acid modulator is preferablywithin about 0.1 to about 250 mg, more preferably within about 1 toabout 100 mg, such as within about 1 to about 5 mg, such as within about1 to about 10 mg, such as within about 1 to about 15 mg, or such aswithin about 1 to about 20 mg.

In another aspect, the invention relates to a compound of formula (I)

wherein

-   -   R¹ and R² are each independently C₁₋₄ alkyl;    -   R³ is independently selected from the group consisting of        hydrogen, halogen, hydroxy, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄        alkoxy, C₁₋₄ haloalkoxy, cyano, nitro, amino, N—(C₁₋₄        alkyl)amino, N,N-di(C₁₋₄ alkyl)amino, and N-(aryl-C₁₋₄        alkyl)amino;    -   n is an integer 1, 2 or 3;    -   R⁴ is selected from the group consisting of hydrogen, halogen,        hydroxy, cyano, C₁₋₄ alkyl, C₃₋₆ cycloalkyl, C₁₋₄ alkoxy, C₃₋₆        cycloalkyloxy, C₁₋₄ alkylthio, C₃₋₆ cycloalkylthio, amino,        N—(C₁₋₄ alkyl)amino and N,N-di(C₁₋₄ alkyl)amino;

or a pharmaceutically acceptable salt thereof, for use as a medicament.

In another aspect, the invention relates to a compound of formula (I),or a pharmaceutically acceptable salt thereof, for use in the treatmentor prevention of any of the diseases recited herein.

The invention also relates to the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment or prevention of any of the diseasesrecited herein. The invention also relates to a method of treating orpreventing any of the diseases recited herein in a subject, such as man,comprising administering to the subject in need of such treatment orprevention a therapeutically effective amount of a compound of formula(I), or a pharmaceutically acceptable salt thereof.

Combination Therapy

In one aspect of the invention, the compounds of formula (I), orpharmaceutically acceptable salts thereof, are administered incombination with at least one other therapeutically active agent, suchas with one, two, three or more other therapeutically active agents. Thecompound of formula (I), or a pharmaceutically acceptable salt thereof,and the at least one other therapeutically active agent may beadministered simultaneously, sequentially or separately. Therapeuticallyactive agents that are suitable for combination with the compounds offormula (I) include, but are not limited to, known active agents thatare useful in the treatment of any of the aforementioned conditions,disorders and diseases.

In one embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anotherASBT inhibitor. Suitable ASBT inhibitors are disclosed in WO 93/16055,WO 94/18183, WO 94/18184, WO 96/05188, WO 96/08484, WO 96/16051, WO97/33882, WO 98/03818, WO 98/07449, WO 98/40375, WO 99/35135, WO99/64409, WO 99/64410, WO 00/47568, WO 00/61568, WO 00/38725, WO00/38726, WO 00/38727, WO 00/38728, WO 00/38729, WO 01/66533, WO01/68096, WO 02/32428, WO 02/50051, WO 03/020710, WO 03/022286, WO03/022825, WO 03/022830, WO 03/061663, WO 03/091232, WO 03/106482, WO2004/006899, WO 2004/076430, WO 2007/009655, WO 2007/009656, WO2011/137135, WO 2019/234077, WO 2020/161216, WO 2020/161217, DE19825804, EP 864582, EP489423, EP549967, EP573848, EP624593, EP624594,EP624595, EP624596, EP0864582, EP 1173205, EP 1535913 and EP 3210977,all of which are incorporated herein by reference in their entireties.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a bileacid binder (also referred to as a bile acid sequestrant, or a resin),such as colesevelam, cholestyramine or cholestipol. In a preferredembodiment of such a combination, the bile acid binder is formulated forcolon release. Examples of such formulations are disclosed in e.g. WO2017/138877, WO 2017/138878, WO 2019/032026 and WO 2019/032027, all ofwhich are incorporated herein by reference in their entireties.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a DPP-IVinhibitor, including gliptins such as sitagliptin, vildagliptin,saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin,alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin anddutogliptin, or a pharmaceutically acceptable salt thereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an HMGCoA reductase inhibitor, such as fluvastatin, lovastatin, pravastatin,simvastatin, atorvastatin, pitavastatin cerivastatin, mevastatin,rosuvastatin, bervastatin or dalvastatin, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with acholesterol absorption inhibitor such as ezetimibe, or apharmaceutically acceptable salt thereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a PPARalpha agonist, including fibrates such as clofibrate, bezafibrate,ciprofibrate, clinofribrate, clofibride, fenofibrate, gemfibrozil,ronifibrate and simfribrate, or a pharmaceutically acceptable saltthereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a PPARgamma agonist, including thiazolidinediones such as pioglitazone,rosiglitazone and lobeglitazone, or a pharmaceutically acceptable saltthereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a dualPPAR alpha/gamma agonist, including glitazars such as saroglitazar,aleglitazar, muraglitazar or tesaglitazar, or a pharmaceuticallyacceptable salt thereof.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a dualPPAR alpha/delta agonist, such as elafibranor.

In yet another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a panPPAR agonist (i.e. a PPAR agonist that has activity across all subtypes:α, γ and δ), such as IVA337.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with afarnesoid X receptor (FXR) modulators, including FXR agonists such ascafestol, chenodeoxycholic acid, 6α-ethyl-chenodeoxycholic acid(obeticholic acid; INT-747), fexaramine, tropifexor, cilofexor andMET409.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a TGR5receptor modulator, including TGR5 agonists such as6α-ethyl-23(S)-methylcholic acid (INT-777).

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a dualFXR/TGR5 agonist such as INT-767.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination withursodeoxycholic acid (UDCA). In yet another embodiment, compounds offormula (I), or pharmaceutically acceptable salts thereof, areadministered in combination with nor-ursodeoxycholic acid (nor-UDCA).

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an FGF19modulator, such as NGM282.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an FGF21agonist, such as BMS-986036.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anintegrin inhibitor, such as PLN-74809 and PLN-1474.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aCCR2/CCR5 inhibitor, such as cenicriviroc.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a caspaseprotease inhibitor, such as emricasan.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with agalectin-3 inhibitor, such as GR-MD-02.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with astearoyl-CoA desaturase (SCD) Inhibitor, such as aramchol (arachidylamido cholanoic acid).

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anapoptosis signal-regulating kinase 1 (ASK1) inhibitor, such asselonsertib.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an LOXL2inhibitor, such as simtuzumab.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an ACCinhibitor, such as GS-0976.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a thyroidhormone receptor-β agonist, such as MGL3196.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a GLP-1agonist such as liraglutide.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a dualglucagon-like peptide and glucagon receptor agonists, such as SAR425899.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with amitochondrial pyruvate carrier inhibitor, such as MSDC-0602K.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with ananti-oxidant agent, such as vitamin E.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an SGLT1inhibitor, an SGLT2 inhibitor or a dual SGLT1 and SGLT2 inhibitor.Examples of such compounds are dapagliflozin, sotagliflozin,canagliflozin, empagliflozin, LIK066 and SGL5213.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with adiacylglycerol O-Acyltransferase 2 (DGAT2) inhibitor, such as DGAT2RXand PF-06865571.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a fattyacid synthase (FASN) Inhibitor, such as TVB-2640.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anAMP-activated protein kinase (AMPK) activator, such as PXL-770.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aglucocorticoid receptor antagonist (GR), a mineralocorticoid receptorantagonist (MR), or a dual GR/MR antagonist. Examples of such compoundsare MT-3995 and CORT-118335.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with acannabinoid receptor 1 (CB1) antagonist, such as IM102.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a Klothop(KLB) and fibroblast growth factor receptor (FGFR) activator, such asMK-3655 (previously known as NGM-313).

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with achemokine (c-c motif) ligand 24 (CCL24) inhibitor, such as CM101.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an A3antagonist, such as PBF-1650.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a P2x7receptor antagonist, such as SGM 1019.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with P2Y13receptor agonists, such as CER-209.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with asulfated oxysterol, such as Dur-928.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aleukotriene D4 (LTD4) receptor antagonist, such as MN-001.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a type 1natural killer T cell (NKT1) inhibitor, such as GRI-0621.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with ananti-lipopolysaccharide (LPS) compound, such as IMM-124E.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a VAP1inhibitor, such as B11467335.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with an A3adenosine receptor agonist, such as CF-102.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a SIRT-1activator, such as NS-20.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anicotinic acid receptor 1 agonist, such as ARI-3037MO.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a TLR4antagonist, such as JKB-121.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aketohexokinase inhibitor, such as PF-06835919.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anadiponectin receptor agonist, such as ADP-335.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with anautotaxin inhibitor, such as PAT-505 and PF8380.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with achemokine (c-c motif) receptor 3 (CCR3) antagonist, such asbertilimumab.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with achloride channel stimulator, such as cobiprostone and lubiprostone.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a heatshock protein 47 (HSP47) inhibitor, such as ND-L02-s0201.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a sterolregulatory element-binding protein (SREBP) transcription factorinhibitor, such as CAT-2003 and MDV-4463.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with abiguanidine, such as metformin.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with insulin.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aglycogen phosphorylase inhibitor and/or a glucose-6-phosphataseinhibitor.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with asulfonylurea, such as glipizid, glibenklamid and glimepirid.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with ameglitinide, such as repaglinide, nateglinide and ormiglitinide.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with aglucosidase inhibitor, such as acarbose or miglitol.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with asqualene synthase inhibitor, such as TAK-475.

In another embodiment, compounds of formula (I), or pharmaceuticallyacceptable salts thereof, are administered in combination with a PTPB1inhibitor, such as trodusquemine, ertiprotafib, JTT-551 and claramine.

Preparation of Compounds

The compounds of the invention can be prepared as a free acid or apharmaceutically acceptable salt thereof by the processes describedbelow. Throughout the following description of such processes it isunderstood that, where appropriate, suitable protecting groups will beadded to, and subsequently removed from the various reactants andintermediates in a manner that will be readily understood by one skilledin the art of organic synthesis. Conventional procedures for using suchprotecting groups as well as examples of suitable protecting groups arefor example described in Greene's Protective Groups in Organic Synthesisby P. G. M Wutz and T. W. Greene, 4th Edition, John Wiley & Sons,Hoboken, 2006.

GENERAL METHODS

All solvents used were of analytical grade. Commercially availableanhydrous solvents were routinely used for reactions. Starting materialswere available from commercial sources or prepared according toliterature procedures. Room temperature refers to 20-25° C. Solventmixture compositions are given as volume percentages or volume ratios.

LCMS:

Instrument name: Agilent 1290 infinity II.

Method A: Mobile phase: A: 0.1% HCOOH in H₂O: ACN (95:5), B: ACN; flowrate: 1.5 mL/min; column: ZORBAX XDB C-18 (50×4.6 mm) 3.5 μM.

Method B: Mobile phase: A: 10 mM NH₄HCO₃ in water, B: ACN; flow rate:1.2 mL/min; column: XBridge C8 (50×4.6 mm), 3.5 μM.

Method C: Mobile phase: A: 0.1% HCOOH in water: ACN (95:5), B: ACN; flowrate: 1.5 mL/min; column: ATLANTIS dC18 (50×4.6 mm), 5 μM.

Method D: Mobile phase: A: 10 mM NH₄OAc in water, B: ACN; flow rate: 1.2mL/min; column: Zorbax Extend C18 (50×4.6 mm) 5 μM.

Method E: Mobile Phase: A: 0.1% TFA in water: ACN (95:5), B: 0.1% TFA inACN; flow rate: 1.5 mL/min; Column: XBridge C8 (50×4.6 mm), 3.5 μM.

UPLC:

Instrument name: waters Acquity I Class

Method A: Mobile Phase: A: 0.1% HCOOH in water, B: 0.1% HCOOH in ACN;Flow Rate: 0.8 mL/min; Column: Acquity UPLC HSS T3 (2.1×50) mm; 1.8 μm.

HPLC:

Instrument name: Agilent 1260 Infinity II series instruments as followedusing % with UV detection (maxplot).

Method A: Mobile phase: A: 10 mM NH₄HCO₃ in water, B: ACN; flow rate:1.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 μm).

Method B: Mobile phase: A: 0.1% TFA in water, B: 0.1% TFA in ACN; flowrate: 2.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 am).

Method C: Mobile phase: A: 10 mM NH₄OAc in milli-q water, B: ACN; flowrate: 1.0 ml/min; column: Phenomenex Gemini C18 (150×4.6 mm, 3.0 am).

Chiral SFC:

Instrument name: PIC SFC 10 (analytical)

Ratio between CO₂ and co-solvent is ranging between 60:40 and 80:20

Method A: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: YMC Amylose-SA (250×4.6 mm, 5 μm).

Method B: Mobile Phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: Chiralpak AD-H (250×4.6 mm, 5 μm).

Method C: Mobile Phase: 20 mM ammonia in methanol; flow rate: 3 mL/min;column: YMC Cellulose-SC (250×4.6 mm, 5 μm).

Method D: Mobile Phase: methanol; flow rate: 3 mL/min; column: Lux A1(250×4.6 mm, 5 m).

Method E: Mobile Phase: 0.5% isopropylamine in methanol; flow rate: 5mL/min; column: Lux C4.

Method F: Mobile Phase: 0.5% isopropylamine in methanol; flow rate: 3mL/min; column: YMC Cellulose-SC.

Method G: Mobile Phase: 0.5% isopropylamine in methanol; flow rate: 3mL/min; column: Lux A1.

Method H: Mobile Phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: Lux A1 (250×4.6 mm, 5 μm).

Method I: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3mL/min; column: Chiral CCS (250×4.6 mm, 5 μm).

Method J: Mobile Phase: 0.5% isopropylamine in IPA; flow rate: 5 mL/min;column: YMC Cellulose-SC AD-H (250×4.6 mm, 5 μm).

Prep-HPLC:

Instrument name: Agilent 1290 Infinity II

Method A: Mobile phase: A: 0.1% TFA in water; Mobile phase; B: 0.1% TFAin ACN; flow rate: 2.0 mL/min; Column: X-Bridge C8 (50×4.6 mm, 3.5 μM).

Method B: Mobile phase: A: 10 mM NH₄OAc in water; B: ACN; flow rate: 35mL/min; column: X select C18 (30×150 mm, 5 μm).

Method C: Mobile phase: A: 10 mM NH₄HCO₃ in water; B: ACN; flow rate:1.0 mL/min; column: XBridge C8 (50×4.6 mm, 3.5 am).

Method D: Mobile phase: A: 0.1% HCOOH in water; B: ACN; flow rate: 1.0mL/min; column: X-select C18 (30×150 mm, 5 μm).

Chiral Preparative SFC:

Instrument name: PIC SFC 100 and PSC SFC 400

Ratio between CO₂ and co-solvent is ranging between 60:40 and 80:20

Method A: Mobile phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: YMC Amylose-SA (250×30 mm, 5 μm).

Method B: Mobile Phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: Chiralpak AD-H (250×30 mm, 5 μm).

Method C: Mobile phase: 20 mM ammonia in methanol; flow rate: 3 mL/min;column: YMC Cellulose-SC (250×30 mm, 5 μm).

Method D: Mobile phase: methanol; flow rate: 3 mL/min; column: ChiralCCS (250×30 mm, 5 μm).

Method E: Mobile phase: methanol; flow rate: 3 mL/min; column: Lux A1(250×30 mm, 5 μm).

Method F: Mobile Phase: 0.5% isopropylamine in IPA; flow rate: 3 mL/min;column: Lux A1 (250×30 mm, 5 μm).

Method G: Mobile phase: 0.5% isopropylamine in methanol; flow rate: 3mL/min; column: Chiral CCS (250×30 mm, 5 μm).

Abbreviations

ACN acetonitrile

DCM dichloromethane

DMF dimethylformamide

IPA isopropyl alcohol

LCMS liquid chromatography-mass spectrometry

HPLC high-performance liquid chromatography

PE petroleum ether

SFC supercritical fluid chromatography

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

UPLC ultra performance liquid chromatography

The invention will now be described by the following examples which donot limit the invention in any respect. All cited documents andreferences are incorporated by reference.

EXAMPLES Intermediate 1 Ethyl 2-aminobutanoate hydrochloride

To a stirred solution of 2-aminobutanoic acid (100 g, 0.97 mol) inethanol (750 mL), thionyl chloride (78 mL, 1.07 mol) was added at 0° C.The reaction mixture was then heated for 16 hours at 80° C. Aftercompletion of the reaction, the reaction mixture was concentrated undervacuum to afford the crude title compound which was used as such for thenext step without any further purification.

Yield: 93% (152 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): δ 8.66 (bs, 3H), 4.25-4.16 (m, 2H), 3.98-3.85(m, 1H), 1.84 (t, J=7.2 Hz, 2H), 1.23 (t, J=6.8 Hz, 3H), 0.92 (t, J=7.6Hz, 3H).

Intermediate 2 Ethyl (E)-2-(benzylideneamino) butanoate

To a stirred solution of ethyl 2-aminobutanoate hydrochloride(Intermediate 1; 152 g, 0.91 mol) in DCM (900 mL), triethyl amine (152mL, 1.09 mol) was added at 0° C. over a period of 30 minutes. Magnesiumsulfate (98 g, 0.82 mol) was added portionwise to the reaction mixtureat 0° C. Benzaldehyde (84 mL, 0.82 mol) was then added to the reactionmixture at 0° C. over a period of 20 minutes and the reaction mixturewas stirred for 16 hours at room temperature. After completion of thereaction (monitored by TLC), the reaction mixture was filtered throughcelite and the filtrate was concentrated under vacuum. The resultingcrude was dissolved in petroleum ether (1000 mL) and again filteredthrough celite. The filtrate was then concentrated under vacuum toafford the title compound. This crude material was forwarded as such tothe next step without any further purification. Yield: 90% (180 g, palebrown liquid).

¹H NMR (400 MHz, DMSO-d₆): δ 8.40 (s, 1H), 7.79-7.76 (m, 2H), 7.49-7.47(m, 3H), 4.16-4.10 (m, 2H), 3.98-3.95 (m, 1H), 1.92-1.89 (m, 1H),1.79-1.74 (m, 1H), 1.19 (t, J=7.2 Hz, 3H), 0.85 (t, J=7.2 Hz, 3H).

Intermediate 3 Ethyl (E)-2-(benzylideneamino)-2-ethylhexanoate

To a stirred solution of NaH (60%; 32.8 g, 0.82 mol) in DMF 100 mL) at0° C., ethyl (E)-2-(benzylideneamino)butanoate (Intermediate 2; 180 g,0.82 mol) in DMF (800 mL) was slowly added over a period of 30 minutes.The reaction mixture was then stirred for 1.5 hours at room temperature.n-Butyl iodide (93 mL, 0.82 mol) was added to the reaction mixture at 0°C. and the mixture was stirred for 1 hour at room temperature. Aftercompletion of the reaction (monitored by TLC), the reaction mixture wasquenched with 2-propanol (100 mL) at 0° C. and then diluted with water(1000 mL). The aqueous layer was extracted with petroleum ether (1000mL). The organic layer was washed with brine (200 mL) and dried overanhydrous Na₂SO₄. The organic part was concentrated under vacuum and theresulting crude material was forwarded as such to the next step withoutany further purification. Yield: 88% (200 g, yellow liquid).

¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (s, 1H), 7.80-7.77 (m, 2H), 7.47-7.44(m, 3H), 4.16 (q, J=7.0 Hz, 2H), 2.51-1.79 (m, 4H), 1.31-1.18 (m, 7H),0.88-0.84 (m, 6H).

Intermediate 4 Ethyl 2-amino-2-ethylhexanoate

To a stirred solution of ethyl (E)-2-(benzylideneamino)-2-ethylhexanoate(Intermediate 3; 200 g, 0.73 mol) in petroleum ether (500 mL), diluteHCl (1000 mL, 1.5 N) was added at 0° C. and the reaction mixture wasstirred vigorously for 16 hours at room temperature. After completion ofthe reaction (monitored by TLC), the organic layer was separated and theaqueous layer was washed with EtOAc (2×100 mL). The aqueous layer wasthen basified (pH 8.5) by using solid sodium bicarbonate (200 g) andextracted with EtOAc (2×200 mL). The organic layer was washed with water(2×15 mL). The combined organic part was dried over anhydrous Na₂SO₄ andconcentrated under vacuum to afford the title compound. The crudematerial was forwarded as such to the next step without any furtherpurification. Yield: 80% (110 g, pale yellow liquid).

¹H NMR (400 MHz, DMSO-d₆): δ 4.08 (q, J=7.1 Hz, 2H), 1.68-1.00 (m, 13H),0.85 (t, J=7.2 Hz, 3H), 0.77 (t, J=7.4 Hz, 3H).

Intermediate 5 2-Amino-2-ethyl-N-phenylhexanamide

To a stirred solution of aniline (48.3 mL, 534 mmol) in THF (250 mL) at−78° C., n-BuLi (2.6M in hexanes; 205 mL, 534 mmol) was added dropwiseover a period of 30 minutes, and the reaction mixture was stirred for 45minutes at −25° C. to −30° C. Then ethyl 2-amino-2-ethylhexanoate(Intermediate 4; 50 g, 267 mmol) in THF (250 mL) was added to thereaction mixture at −78° C. and the reaction mixture was stirred for 2hours at −78° C. After completion of the reaction (monitored by TLC),the reaction mixture was quenched with water (500 mL) at −78° C. Thereaction mixture was extracted with EtOAc (2×250 mL) and the organiclayer was washed with water (2×15 mL). The organic part was dried overanhydrous Na₂SO₄ and concentrated under vacuum to afford the titlecompound as crude. The crude product was dissolved in petroleum ether(1000 mL). The organic part was washed with 30% methanol in water (2×250mL) and dried over anhydrous Na₂SO₄. The organic part was concentratedunder vacuum and the resulting crude was forwarded as such to the nextstep without any further purification. Yield: 66 g (crude, brownliquid).

¹H NMR (400 MHz, DMSO-d₆): δ 7.64 (d, J=8.4 Hz, 2H), 7.30 (t, J=7.4 Hz,2H), 7.05 (t, J=7.4 Hz, 1H), 6.55 (d, J=8.5 Hz, 1H), 1.76-1.07 (m, 10H),0.86-0.77 (m, 6H).

Intermediate 6 2-Ethyl-N1-phenylhexane-1,2-diamine

To a stirred solution of 2-amino-2-ethyl-N-phenylhexanamide(Intermediate 5; 66 g, 0.28 mol) in THF (600 mL), borane dimethylsulfide(2M in THF, 253 mL, 0.51 mol) was added at 0° C. and the reactionmixture was heated for 16 hours at 70° C. After completion of thereaction (monitored by TLC), the reaction mixture was quenched withmethanol (300 mL) at 0° C. The reaction mixture was then heated for 2hours at 70° C. The reaction mixture was concentrated under vacuum andthe obtained residue was dissolved in EtOAc (1000 mL). The organic layerwas washed with water (2×150 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The resulting crude was purified by Isoleracolumn chromatography (eluent: 40% EtOAc in hexane; silica gel: 230-400mesh) to afford the title compound. Yield: 82% (50 g, brown liquid).

¹H NMR (400 MHz, DMSO-d₆): δ 7.04 (t, J=7.2 Hz, 2H), 6.61 (d, J=8.4 Hz,2H), 6.49 (t, J=7.2 Hz, 1H), 5.15 (t, J=4.8 Hz, 1H), 2.79 (d, J=5.6 Hz,2H), 1.39-1.17 (m, 10H), 0.88-0.79 (m, 6H).

Intermediate 7 1,2-Bis(2,4-dibromo-5-methoxyphenyl)disulfane

To a stirred solution of 3-methoxybenzenethiol (100 g, 0.7 mol) inmethanol (1000 mL), bromine (73 mL, 1.4 mol) was added dropwise at 0° C.and the reaction mixture was stirred for 24 hours at room temperature.The reaction mixture was evaporated under vacuum and the obtained crudewas diluted with EtOAc (2000 mL) and washed with water (2×500 mL). Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated undervacuum. The resulting crude was dissolved in glacial acetic acid (600mL), bromine (20 mL) was added dropwise at room temperature and thereaction mixture was stirred for 2 hours at room temperature. Theobtained solid was filtered off, triturated with DCM and dried undervacuum to afford the pure title compound. Yield: 37% (78 g, whitesolid).

¹H NMR (400 MHz, DMSO-d₆): δ 7.69 (s, 2H), 7.17 (s, 2H), 3.84 (s, 6H).

Intermediate 8 2,4-Dibromo-5-methoxybenzenesulfonyl chloride

To a stirred suspension of 1,2-bis(2,4-dibromo-5-methoxyphenyldisulfane(Intermediate 7; 20.0 g, 33.67 mmol) and potassium nitrate (17.02 g,168.35 mmol) in acetonitrile (200 mL) was dropwise added sulfurylchloride (13.6 mL, 168.35 mmol) at 0° C. The reaction mixture wasstirred for 24 hours at room temperature. After completion of thereaction (monitored by TLC), the reaction mixture was poured intocrushed ice and the solid obtained was filtered off. The solid waswashed with water and dried under vacuum to afford the pure titlecompound. Yield: 91% (22.5 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): δ 8.05 (s, 1H), 7.66 (s, 1H), 4.01 (s, 3H).

Intermediate 92,4-Dibromo-5-methoxy-N-(3-((phenylamino)methyl)heptan-3-yl)benzenesulfonamide

To a stirred solution of 2-ethyl-N1-phenylhexane-1,2-diamine(Intermediate 6; 4.9 g, 22.34 mmol) in THF (10 mL) were added2,4-dibromo-5-methoxybenzenesulfonyl chloride (Intermediate 8; 10.5 g,28.91 mmol) and triethylamine (9.3 mL, 67.02 mmol) at 0° C. and thereaction mixture was stirred for 16 hours at room temperature. Aftercompletion of the reaction (monitored by TLC), the reaction mixture wasdiluted with EtOAc (50 mL). The organic layer was washed with water(2×15 mL) and dried over anhydrous Na₂SO₄. The organic part wasconcentrated under vacuum and the resulting crude was purified byIsolera column chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400mesh) to afford the title compound. Yield: 59% (7.2 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): δ 8.01 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H),7.03 (t, J=8.1 Hz, 2H), 6.54-6.46 (m, 3H), 4.80 (t, J=5.1 Hz, 1H), 3.86(s, 3H), 3.07-2.96 (m, 2H), 1.66-1.41 (m, 4H), 1.15-0.95 (m, 4H),0.78-0.69 (m, 6H).

Intermediate 107-Bromo-3-butyl-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide

To a stirred solution of2,4-dibromo-5-methoxy-N-(3-((phenylamino)methyl)heptan-3-yl)benzenesulfonamide(Intermediate 9; 7.2 g, 13.1 mmol) in DMF (50 mL) were added potassiumcarbonate (3.62 g, 26.2 mmol) and copper powder (834 mg, 13.1 mmol) andthe reaction mixture was heated for 24 hours at 150° C. After completionof the reaction (monitored by TLC), the reaction mixture was filteredthrough celite and washed with EtOAc (25 mL). The filtrate part wasconcentrated under vacuum and the resulting crude was purified byIsolera column chromatography (eluent: 20% EtOAc/PE; silica gel: 230-400mesh) to afford the title compound. Yield: 83% (5.1 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): 7.43-7.30 (m, 4H), 7.15-7.13 (m, 2H),7.03-7.01 (m, 2H), 4.00-3.60 (m, 5H), 1.62-1.34 (m, 4H), 1.08-0.95 (m,4H), 0.74-0.71 (m, 6H). LCMS: (Method A) 467.0 (M⁺), Rt. 3.06 min,95.31% (max).

Intermediate 117-Bromo-3-butyl-3-ethyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide

To a stirred solution of7-bromo-3-butyl-3-ethyl-8-methoxy-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide (Intermediate 10; 20.0 g, 42.7 mmol) inN-methyl-2-pyrrolidone (100 mL) were added Cs₂CO₃ (27.8 g, 85.5 mmol)and p-methoxybenzyl bromide (7.98 mL, 39.5 mmol) at 0° C. and thereaction mixture was stirred for 1 hour at room temperature. Aftercompletion of the reaction (monitored by TLC), the reaction mixture wasdiluted with EtOAc (200 mL) and the organic layer was washed with water(2×50 mL). The organic part was dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The resulting crude was purified by Isoleracolumn chromatography (eluent: 10% EtOAc/PE; silica gel: 230-400 mesh)to afford the title compound. Yield: 64% (16 g, white solid).

LCMS: (Method A) 587.2 (M⁺), Rt. 3.51 min, 92.94% (max).

Intermediate 123-Butyl-3-ethyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide

To a stirred solution of7-bromo-3-butyl-3-ethyl-8-methoxy-2-(4-methoxybenzyl)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide (Intermediate 11; 16.0 g, 27.2 mmol) in DMF (120 mL), sodiumthiomethoxide (9.5 g, 136.1 mmol) was added and the reaction mixture washeated for 16 hours at 60° C. After completion of reaction (monitored byLCMS), the reaction mixture was diluted with EtOAc (200 mL) and theorganic layer was washed with water (2×50 mL). The organic part wasdried over anhydrous Na₂SO₄, then concentrated under vacuum and theresulting crude was purified by Isolera column chromatography (eluent:10% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound.Yield: 65% (9.2 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): 10.37 (bs, 1H), 7.31-7.22 (m, 5H), 7.01-6.65(m, 6H), 4.32-4.13 (m, 2H), 4.10-3.90 (m, 2H), 3.74 (s, 3H), 2.15 (s,3H), 1.62-1.34 (m, 4H), 1.08-0.98 (m, 4H), 0.74-0.65 (m, 6H). LCMS:(Method E) 541.2 (M⁺+H), Rt. 2.86 min, 93.67% (max).

Intermediate 13 tert-Butyl2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetate

To a solution of3-butyl-3-ethyl-8-hydroxy-2-(4-methoxybenzyl)-7-(methylthio)-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine1,1-dioxide (Intermediate 12; 1 g, 1.85 mmol) in DMF (5 mL), dry K₂CO₃(0.51 g, 3.70 mmol) and tert-butyl bromoacetate (0.25 mL, 2.77 mmol)were added and the reaction mixture was heated for 16 hours at 100° C.After completion of the reaction (monitored by TLC), the reactionmixture was diluted with EtOAc (20 mL). The organic layer was washedwith water (15 mL) and brine (15 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The resulting crude material was purified byIsolera column chromatography (eluent: 15% EtOAc/PE; silica gel: 230-400mesh) to afford the title compound. Yield: 66% (0.8 g, white solid).

¹H NMR (400 MHz, DMSO-d₆): 7.29-7.27 (m, 4H), 7.11-6.96 (m, 4H), 6.88(d, J=7.8 Hz, 2H), 6.81 (s, 1H), 3.31 (s, 2H), 4.81 (s, 2H), 4.32 (s,2H), 3.75 (s, 3H), 2.15 (s, 3H), 1.40 (s, 13H), 1.11-0.81 (m, 4H),0.78-0.55 (m, 6H). LCMS: (Method A) 655.3 (M⁺+H), Rt. 3.51 min, 90.95%(max).

Intermediate 14 tert-Butyl(S)-2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetateand tert-butyl(R)-2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetate

The two enantiomers of racemic tert-butyl2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetate(0.52 g, 0.79 mmol) were separated by chiral SFC (method H). Thematerial was concentrated under vacuum at 50° C. The first elutingfraction corresponded to enantiomer 1 and the second eluting fractioncorresponded to enantiomer 2. The absolute configuration of the twoenantiomers is not known.

Enantiomer 1: Yield: 38% (200 mg, white solid). Chiral SFC: (Method J)Rt. 3.13 min, 99.90% (Max).

Enantiomer 2: Yield: 42% (220 mg, white solid). LCMS: (Method E) 654.8(M⁺+H), Rt. 3.55 min, 99.21% (Max). HPLC: (Method B) Rt. 7.12 min,99.02% (Max). Chiral SFC: (Method J) Rt. 3.84 min, 98.09% (Max).

Example 12-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiadiazepin-8-yl)oxy)aceticAcid

To a stirred solution of tert-butyl2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)acetate(Intermediate 13; 0.8 g, 1.22 mmol) in DCM (10 mL) at 0° C., TFA (4 mL)and triethylsilane (4 mL) were added and the reaction mixture wasstirred for 4 hours at room temperature. After completion of thereaction (monitored by TLC), the reaction mixture was poured intoice-cold water and the aqueous layer was extracted with DCM (2×10 mL).The combined organic layer was washed with ice-cold water (20 mL) andbrine (20 mL), dried over anhydrous Na₂SO₄ and concentrated undervacuum. The resulting crude material was purified by Prep HPLC (MethodD) to afford the title compound. Yield: 20% (0.12 g, off-white solid).

¹H NMR (400 MHz, DMSO-d₆): δ13.18 (s, 1H), 7.27 (t, J=8.0 Hz, 3H),7.18-7.04 (m, 3H), 6.95-6.93 (m, 1H), 6.60 (s, 1H), 4.77 (s, 2H), 3.82(s, 2H), 2.12 (s, 3H), 1.62-1.52 (m, 2H), 1.49-1.43 (m, 2H), 1.40-1.35(m, 1H), 1.24-0.97 (m, 3H), 0.76-0.72 (m, 6H). LCMS: (Method A) 479.1(M⁺+H), Rt. 2.65 min, 99.44% (max). HPLC: (Method B) Rt. 5.58 min,97.46% (Max).

Examples 2 and 3(S)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticAcid and(R)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticAcid

To a solution of enantiomer 1 of tert-butyl2-((3-butyl-3-ethyl-2-(4-methoxybenzyl)-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiadiazepin-8-yl)oxy)acetate(Intermediate 14; 0.20 g, 0.33 mmol) in toluene (6 mL), triphenylamine(0.16 g, 0.67 mmol) was added and the reaction mixture was cooled to 0°C. Then TFA (0.77 g, 6.72 mmol) was added and the reaction mixture wasstirred for 16 h at room temperature. After completion of the reaction(monitored by TLC), the reaction mixture was diluted with EtOAc (15 mL),and the organic layer was washed with water (2×10 mL) and brine (10 mL).The organic part was dried over anhydrous Na₂SO₄ and concentrated undervacuum. The resulting crude material was purified by Isolera columnchromatography (eluent: 30% EtOAc/PE; silica gel: 230-400 mesh) toafford enantiomer 1 of the title compound.

Enantiomer 2 of the title compound was obtained following the sameprocedure, starting from 0.22 g of enantiomer 2 of Intermediate 14. Theabsolute configuration of the two enantiomers is not known.

Enantiomer 1: Yield: 43% (70 mg, white solid). ¹H NMR (400 MHz,DMSO-d₆): δ 13.14 (bs, 1H), 7.29-7.25 (m, 3H), 7.11-7.06 (m, 3H), 6.94(t, J=7.2 Hz, 1H), 6.60 (s, 1H), 4.78 (s, 2H), 3.85 (bs, 2H), 2.12 (s,3H), 1.72-1.58 (m, 1H), 1.57-1.45 (m, 1H), 1.43-1.35 (m, 2H), 1.29-1.14(m, 1H), 1.13-0.88 (m, 3H), 0.74 (t, J=7.2 Hz, 6H). LCMS: (Method E)478.8 (M⁺+H), Rt. 2.92 min, 98.18% (max). HPLC: (Method B) Rt. 5.54 min,97.87% (Max). Chiral SFC: (Method G) Rt. 2.0 min, 99.30% (Max).

Enantiomer 2: Yield: 37% (60 mg, white solid). ¹H NMR (400 MHz,DMSO-d₆): δ 13.10 (bs, 1H), 7.29-7.25 (m, 3H), 7.11-7.06 (m, 3H),6.96-6.94 (m, 1H), 6.60 (s, 1H), 4.78 (s, 2H), 3.80 (bs, 2H), 2.12 (s,3H), 1.72-1.58 (m, 1H), 1.57-1.45 (m, 1H), 1.43-1.35 (m, 2H), 1.29-1.13(m, 1H), 1.13-0.88 (m, 3H), 0.74 (t, J=7.2 Hz, 6H). LCMS: (Method E)478.9 (M⁺+H), Rt. 2.92 min, 98.99% (max), HPLC: (Method B) Rt. 5.53 min,97.81% (Max). Chiral SFC: (Method G) Rt. 1.89 min, 100% (Max).

Biological Assays

IBAT (h/m) Assay Protocol

10,000 cells (Human or Mouse IBAT-overexpressing cells) were seeded in96-wells plate (Corning CLS3809) in 200 μL MEM-alpha medium (Gibco12571-063) supplemented with 10% FBS (Gibco 10438026) containingPuromycin (Gibco A1113803) (10 μg/mL) and incubated at 37° C. in 5% CO₂for 48 hours. After incubation, media was decanted from the wells andcells were washed two times with 300 μL of basal MEM-alpha medium(FBS-free). After decanting basal MEM-alpha medium each time, plateswere tapped against paper towel to ensure maximum removal of residualmedia.

Test inhibitor dilutions (highest test concentration being 10 μM, 3-foldserial dilution, 10 points) prepared in DMSO (Sigma D2650) were added inincubation mix (maintaining 0.2% final DMSO concentration) containing0.25 μM 3H-taurocholic acid (ARC ART-1368) and 5 μM of cold taurocholicacid (Sigma T4009). 50 μL of incubation mix containing test inhibitorswas then added to the wells (in duplicate) and the plates were incubatedfor 20 minutes in a CO₂ incubator at 37° C. After incubation, thereaction was stopped by keeping the plates on ice water mix for 2-3minutes and then the incubation mix was aspirated completely from thewells. The wells were washed two times with 250 μL of chilled unlabeled1 mM taurocholic acid dissolved in HEPES (Gibco 15630080)-buffered (10mM) HBSS (Gibco 14175079) (pH 7.4). The plates were tapped against apaper towel after every wash to ensure maximum removal of blockingbuffer.

100 μL of MicroScint-20 (PerkinElmer 6013621) was added to the wells andkept overnight at room temperature before reading the plates in TopCountNXT™ Microplate Scintillation and Luminescence Counter from PerkinElmerunder 3H Test protocol (set at 120 seconds reading time per well).

LBAT (h/m) Assay Protocol

20,000 cells (Human or Mouse LBAT-overexpressing cells) were seeded in96-wells plate (Corning CLS3809) in 100 μL MEM-alpha medium (Gibco12571-063) supplemented with 10% FBS (Gibco 10438026) containingGeneticin (Gibco 10131-027) (1 mg/mL) and incubated at 37° C. in 5% CO₂for 24 hours. After incubation, media was decanted from the wells andcells were washed two times with 300 μL of basal MEM-alpha medium(FBS-free). After decanting basal MEM-alpha medium each time, plateswere tapped against paper towel to ensure maximum removal of residualmedia.

For human LBAT, incubation mix was prepared by adding test inhibitordilutions (3-fold serial dilution in DMSO (Sigma D2650), 10 points) inMEM-alpha (without FBS) containing 0.3 μM 3H-taurocholic acid (ARCART-1368) and 7.5 μM cold taurocholic acid (Sigma T4009) (maintaining0.2% final DMSO concentration). For mouse LBAT, incubation mix wasprepared by adding test inhibitor dilutions (3-fold serial dilution inDMSO, 10 points) in MEM-alpha (without FBS) containing 0.3 μM3H-taurocholic acid and 25 μM cold taurocholic acid maintaining 0.2%final DMSO concentration). 50 μL of incubation mix containing testinhibitors was then added to the wells (in duplicate) and the plateswere incubated for 20 minutes in a CO₂ incubator at 37° C. Afterincubation, the reaction was stopped by keeping the plates on ice watermix for 2-3 minutes and then the incubation mix was aspirated completelyfrom the wells. The wells were washed two times with 250 μL of chilledunlabeled 1 mM taurocholic acid dissolved in HEPES (Gibco15630080)-buffered (10 mM) HBSS (Gibco 14175079) (pH 7.4). The plateswere tapped against a paper towel after every wash to ensure maximumremoval of blocking buffer.

100 μL of MicroScint-20 (PerkinElmer 6013621) was added to the wells andkept overnight at room temperature before reading the plates in TopCountNXT™ Microplate Scintillation and Luminescence Counter from PerkinElmerunder 3H Test protocol (set at 120 seconds reading time per well, withnormal plate orientation).

Bidirectional Permeability Assay (Caco-2 Cells)

Caco-2 cells (Evotec) were seeded at a density of 70,000 cells/well inMillicell® 24-well cell culture insert plates and maintained in anincubator (37° C., 5% CO₂, 95% RH) for 21 days with media change onalternate days.

Stock solutions (10 mM) of the test compounds, atenolol (lowpermeability marker), propranolol (high permeability marker) and digoxin(substrate for P-gp transport pathway) were prepared indimethylsulfoxide (DMSO). An intermediate stock solution (1 mM) wasprepared by diluting 10 μL of 10 mM master stock solution with 90 μL ofneat DMSO. A working stock solution (10 μM) was prepared by diluting 50μL of 1 mM with 4950 μL of FaSSIF buffer. Post addition of compounds tothe FaSSIF, samples were subjected to sonication for 2 hours, andcentrifuged at 4000 RPM for 30 minutes at 37° C. The 4 mL of resultantsupernatant was directly used in the assay. The final DMSO concentrationin the transport experiments was 1%.

On the day of assay, Caco-2 monolayers were washed twice with transportbuffer (HBSS, pH 7.4) and pre-incubated for 30 min (37° C., 5% CO₂, 95%RH) in an incubator. The electrical resistance of the monolayers wasmeasured with a Millicell®—ERS system. Monolayers with trans-epithelialelectrical resistance (TEER) values greater than 350 ohm·cm² wereselected for the assay.

The assay was conducted in absorptive direction (A2B) and secretory(B2A) directions. Transport experiments were initiated by addition oftransport assay buffer (FaSSIF buffer prepared in HBSS) consisting ofcompounds to the donor compartment (apical chamber A-B; basolateralchamber B-A) in duplicate (n=2) wells. Drug free HBSS buffer (pH 7.4)containing 1% bovine serum albumin (BSA) was introduced to the receiver(A-B-basolateral; B-A-Apical) compartments. The volumes of apical andbasolateral compartments were 0.4 and 0.8 mL, respectively. After addingdosing solution, plates were incubated in an incubator for 120 minutesat 37° C. After 120 minutes, donor and receiver samples were collectedand matrix matched (1:1, 30 μL study sample+30 μL blank buffer) with theopposite buffer. Dosing samples matrix matched (1:1, 30 μL studysample+30 μL blank buffer) with the opposite buffer. Samples wereprocessed by adding acetonitrile containing internal standard (60 μLstudy sample+200 μL acetonitrile containing internalstandard-Tolbutamide, 500 ng/mL). Samples were vortexed and centrifugedat 4000 rpm for 10 minutes. The obtained supernatant (100 μL) wasdiluted with 100 μL of water and transferred to fresh 96 well plates.The concentration of compounds in the samples was analyzed by liquidchromatography tandem mass spectrometry (LC-MS/MS) method usingdiscovery grade bio-analytical method, as applicable.

The mean apparent permeability (P_(app), ×10⁻⁶ cm/sec) of the testcompounds, atenolol, propranolol and digoxin were calculated as follows:

${Papp} = {\frac{dq}{dt} \times \frac{1}{Co} \times \frac{1}{A}}$

where dq/dt=rate of transport (rate of transport of compound in thereceiver compartment), C₀=initial concentration in the donorcompartment, A=surface area of the effective filter membrane.

HepaRG-Based Assay Protocol

A cryopreserved vial of differentiated HepaRG cells (BiopredicInternational HPR116080) is thawed in HepaRG Thawing/Plating/GeneralPurpose Medium (Biopredic International ADD670C) supplemented with 200mM Glutamax (Gibco 35050061) following the protocol provided byBiopredic International. 70,000 cells per well are seeded in 96-wellsplate (Corning CLS3809) in 100 μL of HepaRG Thawing/Plating/GeneralPurpose Medium supplemented with 200 mM Glutamax and incubated at 37° C.in 5% CO₂ for 24 hours. Post incubation, the seeding media is replacedby HepaRG Maintenance/Metabolism Medium (Biopredic InternationalADD620C) and incubated for 6 days, with fresh HepaRGMaintenance/Metabolism Medium replenishment every 48 hours. After 7 daysincubation post seeding, incubation media is decanted from the wells andcells are washed two times with 250 μL of William's E Basal Media (Gibco12551032). After decanting William's E Basal Media each time, plates aretapped against paper towel to ensure maximum removal of residual media.Incubation mix is prepared by adding test inhibitor dilutions (3-foldserial dilution in DMSO (Sigma D2650)) in William's E media (basal)containing 0.3 μM 3H-taurocholic acid (ARC ART-1368) and 7.5 μM coldtaurocholic acid (Sigma T4009) (maintaining 0.2% final DMSOconcentration). 50 μl of incubation mix containing test inhibitors isthen added to the wells (in duplicate) and the plates are incubated for30 minutes in 5% CO₂ incubator at 37° C. After incubation, the reactionis stopped by keeping the plates on ice water mix for 2-3 minutes andthe incubation mix is then aspirated completely from the wells. Thewells are washed two times with 250 μL of chilled unlabeled 1 mMtaurocholic acid dissolved in HEPES (Gibco 15630080)-buffered (10 mM)HBSS (Gibco 14175079) (pH 7.4). The plates are tapped against a papertowel after every wash to ensure maximum removal of blocking buffer.

100 μL of MicroScint-20 (PerkinElmer 6013621) is added to the wells andkept overnight at room temperature before reading the plates in TopCountNXT™ Microplate Scintillation and Luminescence Counter from PerkinElmerunder 3H Test protocol (set at 120 seconds reading time per well, withnormal plate orientation).

Preparation of Test Compound Dilutions

All test compounds were provided in powder form at room temperature. 10mM DMSO stocks of the test compounds were prepared, aliquoted and storedat −20° C. From the 10 mM DMSO stock of the compounds, a 3-fold serialdilution in DMSO was prepared to get a total of 10 dilutions of the testcompounds. 0.5 μL of this dilution in DMSO was added to 250 μL ofFBS-free basal media containing 3H-taurocholic acid and cold taurocholicacid to prepare the incubation mixture.

Bioavailability Studies

Male mice (C57BL/6 or CD1) or Wistar rats of 8-9 weeks old were used.For each test compound, two groups of 3 animals each were used. Onegroup was administered a single intravenous dose of 1 mg/kg (vehicle100% DMSO) through the tail vein and the other group was administered asingle oral dose of 10 mg/kg through gavage needle. The group that wasadministered an oral dose was fasted overnight. Blood samples werecollected after 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours followingintravenous administration, and after 0.25, 0.5, 1, 2, 4, 6, 8 and 24hours following oral administration. Blood samples were taken fromsaphenous vein. 0.2% EDTA was used as the anticoagulant. The sampleswere analyzed by a discovery grade bioanalytical method developed forthe estimation of test compound in plasma, using an LC-MS/MS system.

Results

Biological data for the compounds of the examples is shown in Table 8below.

TABLE 8 Permeability (Caco-2) hLBAT IC₅₀ hIBAT IC₅₀ P_(app) A2B P_(app)B2A Example (nM) (nM) (×10⁻⁶ cm/sec) (×10⁻⁶ cm/sec) 1 8.8 1.1 2.9 2 39795.7 2.52 4.0 3 4998 182

PD Model: Evaluation of Test Compound on Total Bile Acids Levels in MaleC57BL6 Mice.

C57BL/6N Tac mice of 8-9 weeks old are used to study the effect of bileacid modulators on bile acid levels. After completion of quarantine andacclimatization period, animals are randomized based on bodyweight intox experimental groups: (i) vehicle control, and (ii) test compound ymg/kg po once daily. Animals are treated with test compound for 7 days.On day 5 of the study, animals are individually housed in fresh cages.On day 7, feces are collected from each cage, followed by bloodwithdrawal from each animal through retro-orbital route. Animals areeuthanized to collect liver and terminal ileum from each animal forfurther analysis. Bodyweight and food consumption are measured twiceweekly. Serum lipid profiles are analyzed in serum samples of day 7.Total bile acids in serum is measured in the serum samples of day 7.Fecal bile excretion is measured in the fecal sample of day 7. Hepaticexpression of CYP7A1 and SHP are quantified in the liver samples of day7. Liver triglycerides and total cholesterol are analyzed in the liversamples of day 7.

Urine Bile Acid Model: Evaluation of Test Compounds on Urine Bile AcidLevels in Male C57BL/6N Mice.

C57BL/6N Tac mice of 8-9 weeks old are used to study the effect of bileacid modulators on bile acid levels. After completion of quarantine andacclimatization period, animals are randomized based on bodyweight intox experimental groups: (i) vehicle control, and (ii) test compound ymg/kg po once daily. Animals are treated with test compound for 7 days.On day 6 of the study, animals are transferred to a metabolic cage. Onday 7, feces and urine are collected from each metabolic cage, followedby blood withdrawal from each animal through retro-orbital route.Animals are euthanized to collect kidney from each animal for furtheranalysis. Bodyweight is measured twice weekly. Total bile acids in serumis measured in serum samples of day 7. Fecal bile acid excretion ismeasured in the fecal sample of day 7. Urine excretion of bile acids ismeasured in the sample of day 7. Kidney expression of ASBT, OSTa, OSTAband MRP2 is quantified in the samples of day 7.

1. A compound of formula (I)

wherein R¹ and R² are each independently C₁₋₄ alkyl; R³ is independentlyselected from the group consisting of hydrogen, halogen, hydroxy, C₁₋₄alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, cyano, nitro,amino, N—(C₁₋₄ alkyl)amino, N,N-di(C₁₋₄ alkyl)amino, and N-(aryl-C₁₋₄alkyl)amino; n is an integer 1, 2 or 3; and R⁴ is selected from thegroup consisting of hydrogen, halogen, hydroxy, cyano, C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₁₋₄ alkoxy, C₃₋₆ cycloalkyloxy, C₁₋₄ alkylthio, C₃₋₆cycloalkylthio, amino, N—(C₁₋₄ alkyl)amino and N,N-di(C₁₋₄ alkyl)amino;or a pharmaceutically acceptable salt thereof, with the proviso that thecompound is not a compound from the group consisting of2-((3,3-dibutyl-7-methyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticacid;2-((3,3-dibutyl-7-(methylthio)-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticacid; and2-((7-bromo-3,3-dibutyl-5-phenyl-1,1-dioxido-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticacid.
 2. A compound according to claim 1, wherein R¹ is n-butyl.
 3. Acompound according to claim 1, wherein R² is n-butyl.
 4. A compoundaccording to claim 1, wherein R² is ethyl.
 5. A compound according toclaim 1, wherein R³ is independently selected from the group consistingof hydrogen, halogen, hydroxy, cyano, C₁₋₄ haloalkyl, C₁₋₄ alkoxy andC₁₋₄ haloalkoxy.
 6. A compound according to claim 1, wherein R³ isindependently selected from the group consisting of hydrogen, fluoro,chloro, bromo, hydroxy, cyano, trifluoromethyl, methoxy andtrifluoromethoxy.
 7. A compound according to claim 1, wherein R⁴ isselected from the group consisting of halogen, hydroxy, cyano, C₁₋₄alkyl, C₁₋₄ alkoxy, C₁₋₄ alkylthio, amino, N—(C₁₋₄ alkyl)amino andN,N-di(C₁₋₄ alkyl)amino.
 8. A compound according to claim 1, wherein R⁴is selected from the group consisting of fluoro, chloro, bromo, hydroxy,cyano, methyl, methoxy, ethoxy, methylthio, ethylthio, amino,methylamino and dimethylamino.
 9. A compound according to claim 1,selected from the group consisting of2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydrobenzo-1,2,5-thiadiazepin-8-yl)oxy)aceticacid;(S)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticacid; and(R)-2-((3-butyl-3-ethyl-7-(methylthio)-1,1-dioxido-5-phenyl-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepin-8-yl)oxy)aceticacid; or a pharmaceutically acceptable salt thereof.
 10. Apharmaceutical composition comprising a therapeutically effective amountof a compound according to claim 1, or a pharmaceutically acceptablesalt thereof, and one or more pharmaceutically acceptable excipients.11. (canceled)
 12. The method according to claim 17, wherein thecardiovascular disease or disorder of fatty acid metabolism or glucoseutilization disorder is selected from the group consisting of:hypercholesterolemia; a disorder of fatty acid metabolism; type 1 andtype 2 diabetes mellitus; a complication of diabetes.
 13. The methodaccording to claim 18, wherein the gastrointestinal disease or disorderis selected from the group consisting of: constipation; Crohn's disease;primary bile acid malabsorption; irritable bowel syndrome (IBS);inflammatory bowel disease (IBD); ileal inflammation; and reflux diseaseand complications thereof.
 14. The method according to claim 19, whereinthe liver disease or disorder is selected from the group consisting ofan inherited metabolic disorder of the liver; inborn errors of bile acidsynthesis; congenital bile duct anomalies; biliary atresia; post-Kasaibiliary atresia; post-liver transplantation biliary atresia; neonatalhepatitis; neonatal cholestasis; hereditary forms of cholestasis;cerebrotendinous xanthomatosis; a secondary defect of BA synthesis;Zellweger's syndrome; cystic fibrosis-associated liver disease;alpha1-antitrypsin deficiency; Alagilles syndrome (ALGS); Bylersyndrome; a primary defect of bile acid (BA) synthesis; progressivefamilial intrahepatic cholestasis (PFIC); benign recurrent intrahepaticcholestasis (BRIC); autoimmune hepatitis; primary biliary cirrhosis(PBC); liver fibrosis; non-alcoholic fatty liver disease (NAFLD);non-alcoholic steatohepatitis (NASH); portal hypertension; cholestasis;Down syndrome cholestasis; drug-induced cholestasis; intrahepaticcholestasis of pregnancy (jaundice during pregnancy); intrahepaticcholestasis; extrahepatic cholestasis; parenteral nutrition associatedcholestasis (PNAC); low phospholipid-associated cholestasis; lymphedemacholestasis syndrome 1 (LSC1); primary sclerosing cholangitis (PSC);immunoglobulin G4 associated cholangitis; primary biliary cholangitis;cholelithiasis (gall stones); biliary lithiasis; choledocholithiasis;gallstone pancreatitis; Caroli disease; malignancy of bile ducts;malignancy causing obstruction of the biliary tree; biliary strictures;AIDS cholangiopathy; ischemic cholangiopathy; pruritus due tocholestasis or jaundice; pancreatitis; chronic autoimmune liver diseaseleading to progressive cholestasis; hepatic steatosis; alcoholichepatitis; acute fatty liver; fatty liver of pregnancy; drug-inducedhepatitis; iron overload disorders; congenital bile acid synthesisdefect type 1 (BAS type 1); drug-induced liver injury (DILI); hepaticfibrosis; congenital hepatic fibrosis; hepatic cirrhosis; Langerhanscell histiocytosis (LCH); neonatal ichthyosis sclerosing cholangitis(NISCH); erythropoietic protoporphyria (EPP); idiopathic adulthoodductopenia (IAD); idiopathic neonatal hepatitis (INH); non syndromicpaucity of interlobular bile ducts (NS PILBD); North American Indianchildhood cirrhosis (NAIC); hepatic sarcoidosis; amyloidosis;necrotizing enterocolitis; serum bile acid-caused toxicities; polycysticliver disease; viral hepatitis; hepatocellular carcinoma (hepatoma);cholangiocarcinoma; bile acid-related gastrointestinal cancers; andcholestasis caused by tumours and neoplasms of the liver, of the biliarytract and of the pancreas.
 15. The method according to claim 16, whereinthe hyperabsorption syndrome is selected from the group consisting of:abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL);chylomicron retention disease (CRD); and sitosterolemia.
 16. A methodfor treating a disease or disorder in a subject, the method comprisingadministering to the subject a therapeutically effective amount of acompound of formula (I)

wherein R¹ and R² are each independently C₁₋₄ alkyl; R³ is independentlyselected from the group consisting of hydrogen, halogen, hydroxy, C₁₋₄alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkoxy, cyano, nitro,amino, N—(C₁₋₄ alkyl)amino, N,N-di(C₁₋₄ alkyl)amino, and N-(aryl-C₁₋₄alkyl)amino; n is an integer 1, 2 or 3; and R⁴ is selected from thegroup consisting of hydrogen, halogen, hydroxy, cyano, C₁₋₄ alkyl, C₃₋₆cycloalkyl, C₁₋₄ alkoxy, C₃₋₆ cycloalkyloxy, C₁₋₄ alkylthio, C₃₋₆cycloalkylthio, amino, N—(C₁₋₄ alkyl)amino and N,N-di(C₁₋₄ alkyl)amino;or a pharmaceutically acceptable salt thereof, wherein the disease ordisorder is selected from the group consisting of a cardiovasculardisease; a disorder of fatty acid metabolism; a glucose utilizationdisorder; a gastrointestinal disease or disorder; a liver disease ordisorder; a hyperabsorption syndrome; hypervitaminosis andosteopetrosis; hypertension; glomerular hyperfiltration; or pruritus ofrenal failure.
 17. The method according to claim 16, wherein the diseaseor disorder is selected from the group consisting of a cardiovasculardisease, a disorder of fatty acid metabolism, and a glucose utilizationdisorder.
 18. The method according to claim 16, wherein the disease ordisorder is a gastrointestinal disease or disorder.
 19. The methodaccording to claim 16, wherein the disease or disorder is a liverdisease or disorder.
 20. The method according to claim 14, wherein theliver disease or disorder is a cholestatic liver disease.
 21. The methodaccording to claim 20, wherein the liver disease or disorder is selectedfrom the group consisting of: progressive familial intrahepaticcholestasis (PFIC), Alagilles syndrome, biliary atresia, andnon-alcoholic steatohepatitis (NASH).